Stable Isotope Tracer Studies Research Articles

Fate of 15N-enriched cyanobacteria feed for planktivorous fish in an enclosure experiment: a stable isotope tracer study

A tracer experiment using isotope 15N was performed to study the assimilation and retention of nitrogen from feces by two planktivorous fish, silver carp Hypophthalmichthys molitrix and tilapia Oreochromis niloticus, in Lake Taihu (China). Microcystis was enriched with 15N-NH4Cl, lyophilized to produce feed for fish, and traced to establish the fate of feces nitrogen. Samples of organisms and abiotic substances were analyzed for excess 15N and nutrient concentrations in the water column were determined on days 0, 1, 5, 10, 15, and 20. Nutrient concentration analyses indicated that TN and TP were about 4 times higher in the tilapia enclosure than in the silver carp enclosure due to the digestive capacity of Microcystis, which suggests that the ichthyoeutrophication potential of tilapia is greater than that of silver carp. 11.05 % of the 15N was assimilated by the tilapia whereas 3.58 % of the 15N was assimilated by the silver carp, suggesting that tilapia has a higher capacity to assimilate and retain Microcystis nitrogen than silver carp, although the absorptivities of both species were relatively low. At the end of the experiment, 8.48 % of the 15N was detected in sedimentary detritus in the tilapia enclosure, as compared to 6.07 % of the 15N in the silver carp enclosure, which suggests that only a small fraction of the Microcystis-derived nitrogen sank to the bottom. In conclusion, much of the Microcystis-derived nitrogen was neither assimilated by fish nor accumulated on the sediment floor. It presumably floated in the water column, contributing to phytoplankton propagation and thus degrading the water quality.

The Size of the Exchangeable Zinc Pool (EZP) is Related to Age, Body Size, Gender and Plasma Zinc Concentration in Adults

The EZP is the pool of rapidly exchanging, metabolically active zinc (Zn) in the human body. Its size is measured routinely in stable isotope tracer studies of Zn absorption, and it has been given consideration as a potential biomarker of Zn status. Linear regression models were used to investigate relationships of EZP to body size, age, gender, plasma Zn concentration, dietary Zn intake and absorbed Zn in data from studies of healthy adults. EZP size measurements ranged from 75 to 280 mg in subjects between 18 and 83 yrs of age. Age, body weight (or surface area), gender and plasma Zn (in decreasing order of importance) were all significant predictors of EZP in multiple regression models of data from 100 subjects having adequate Zn intake (>7 mg/d) and absorption (> 2.8 mg/d). The models showed EZP size to increase with body weight and plasma Zn, and to decrease with age. The gender difference was largest in young adults (EZP in males was 50 mg larger at 20 yrs of age) and decreased to equality at 80 yrs. Younger subjects with Zn intakes-absorption lower than the ranges mentioned above had smaller EZP sizes on average than model predictions when controlling for age, body weight and gender. No such difference was evident in elderly subjects having lower intakes-absorption. EZP sizes in subjects experiencing severe Zn depletion were consistently lower than the model's 95% prediction intervals. The models have descriptive and predictive utility: contributing to an understanding of the physiological characteristics of the EZP, establishing normal values and ranges for healthy subjects and evaluating the suitability of EZP size as a biomarker of Zn status.

Effect of Error Propagation in Stable Isotope Tracer Studies: An Approach for Estimating Impact on Apparent Biochemical Flux.

Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e., the precursor and product labeling pre- and posttracer administration. This seemingly simple problem has 1 correct solution and 80 erroneous outcomes. In this report, we outline a methodology for evaluating the effect of error propagation on apparent physiological endpoints. We demonstrate examples of how to evaluate the influence of analytical error in case studies concerning lipid and protein synthesis; we have focused on (2)H2O as a tracer and contrast different mass spectrometry platforms including GC-quadrupole-MS, GC-pyrolysis-IRMS, LC-quadrupole-MS, and high-resolution FT-ICR-MS. The method outlined herein can be used to determine how to minimize variations in the apparent biology by altering the dose and/or the type of tracer. Likewise, one can facilitate biological studies by estimating the reduction in the noise of an outcome that is expected for a given increase in the number of replicate injections.

The role of dietary sugars and de novo lipogenesis in non-alcoholic fatty liver disease.

Dietary sugar consumption, in particular sugar-sweetened beverages and the monosaccharide fructose, has been linked to the incidence and severity of non-alcoholic fatty liver disease (NAFLD). Intervention studies in both animals and humans have shown large doses of fructose to be particularly lipogenic. While fructose does stimulate de novo lipogenesis (DNL), stable isotope tracer studies in humans demonstrate quantitatively that the lipogenic effect of fructose is not mediated exclusively by its provision of excess substrates for DNL. The deleterious metabolic effects of high fructose loads appear to be a consequence of altered transcriptional regulatory networks impacting intracellular macronutrient metabolism and altering signaling and inflammatory processes. Uric acid generated by fructose metabolism may also contribute to or exacerbate these effects. Here we review data from human and animal intervention and stable isotope tracer studies relevant to the role of dietary sugars on NAFLD development and progression, in the context of typical sugar consumption patterns and dietary recommendations worldwide. We conclude that the use of hypercaloric, supra-physiological doses in intervention trials has been a major confounding factor and whether or not dietary sugars, including fructose, at typically consumed population levels, effect hepatic lipogenesis and NAFLD pathogenesis in humans independently of excess energy remains unresolved.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.

Glucose metabolism during the early “flow phase” after burn injury

BackgroundBurn injury (BI) is associated with insulin resistance (IR) and hyperglycemia which complicate clinical management. We investigated the impact of BI on glucose metabolism in a rabbit model of BI using a combination of positron emission tomography (PET) and stable isotope studies under euglycemic insulin clamp (EIC) conditions. Materials and methodsTwelve male rabbits were subjected to either full-thickness BI (B) or sham burn. An EIC condition was established by constant infusion of insulin, concomitantly with a variable rate of dextrose infusion 3 d after treatment. PET imaging of the hind limbs was conducted to determine the rates of peripheral O2 and glucose utilization. Each animal also received a primed constant infusion of [6,6-2H2] glucose to determine endogenous glucose production. ResultsThe fasting blood glucose in the burned rabbits was higher than that in the sham group. Under EIC conditions, the sham burn group required more exogenous dextrose than the B group to maintain blood glucose at physiological levels (22.2 ± 2.6 versus 13.3 ± 2.9 mg/min, P < 0.05), indicating a state of IR. PET imaging demonstrated that the rates of O2 consumption and 18F 2-fluoro-2-deoxy-D-glucose utilization by skeletal muscle remained at similar levels in both groups. Hepatic gluconeogenesis determined by the stable isotope tracer study was found significantly increased in the B group. ConclusionsThese findings demonstrated that hyperglycemia and IR develop during the early “flow phase” after BI. Unsuppressed hepatic gluconeogenesis, but not peripheral skeletal muscular utilization of glucose, contributes to hyperglycemia at this stage.

Zinc isotope ratio imaging of rat brain thin sections from stable isotope tracer studies by LA-MC-ICP-MS

Zinc stable isotope tracers (⁶⁷Zn and ⁷⁰Zn) were injected into rats at two different time points to investigate the feasibility of using tracers to study zinc kinetics at the microscale within distinct tissue features. Laser ablation coupled to multi-collector ICP-MS was used to analyse average isotope ratios in liver thin sections and to generate bio-images showing zinc isotope ratio distribution in brain thin sections. Average isotope ratios of all samples from treated animals were found to be statistically different (P < 0.05) from samples from untreated control animals. Furthermore, differing isotope ratios in physiological features of the brain, namely hippocampus, amygdala, cortex and hypothalamus, were identified. This indicates that these regions differ in their zinc metabolism kinetics. While cortex and hypothalamus contain more tracer two days after injection than 14 days after injection, the opposite is true for hippocampus and amygdala. This study showed that stable isotope tracer experiments can be combined with laser ablation MC-ICP-MS to measure trace element kinetics in tissues at a microscale level.

A Stable Isotope Tracer Study of the Influences of Adjacent Land Use and Riparian Condition on Fates of Nitrate in Streams

The influence of land use on potential fates of nitrate (NO3−) in stream ecosystems, ranging from denitrification to storage in organic matter, has not been documented extensively. Here, we describe the Pacific Northwest component of Lotic Intersite Nitrogen eXperiment, phase II (LINX II) to examine how land-use setting influences fates of NO3− in streams. We used 24 h releases of a stable isotope tracer (15NO3-N) in nine streams flowing through forest, agricultural, and urban land uses to quantify NO3− uptake processes. NO3− uptake lengths varied two orders of magnitude (24–4247 m), with uptake rates (6.5–158.1 mg NO3-N m−2 day−1) and uptake velocities (0.1–2.3 mm min−1) falling within the ranges measured in other LINX II regions. Denitrification removed 0–7% of added tracer from our streams. In forest streams, 60.4 to 77.0% of the isotope tracer was exported downstream as NO3−, with 8.0 to 14.8% stored in wood biofilms, epilithon, fine benthic organic matter, and bryophytes. Agricultural and urban streams with streamside forest buffers displayed hydrologic export and organic matter storage of tracer similar to those measured in forest streams. In agricultural and urban streams with a partial or no riparian buffer, less than 1 to 75% of the tracer was exported downstream; much of the remainder was taken up and stored in autotrophic organic matter components with short N turnover times. Our findings suggest restoration and maintenance of riparian forests can help re-establish the natural range of NO3− uptake processes in human-altered streams.

Validation of a single biopsy approach and bolus protein feeding to determine myofibrillar protein synthesis in stable isotope tracer studies in humans

BackgroundMinimizing the number of muscle biopsies has important methodological implications and minimizes subject discomfort during a stable isotope amino acid infusion. We aimed to determine the reliability of obtaining a single muscle biopsy for the calculation of muscle protein fractional synthetic rate (FSR) as well as the amount of incorporation time necessary to obtain that biopsy after initiating a stable isotope infusion (Study 1). The calculation of muscle protein FSR requires tracer steady-state during the stable isotope infusion. Therefore, a second aim was to examine if steady-state conditions are compromised in the precursor pools (plasma free or muscle intracellular [IC]) after ingestion of a tracer enriched protein drink and after resistance exercise (Study 2).MethodsSixteen men (23 ± 3 years; BMI = 23.8 ± 2.2 kg/m2, means ± SD) were randomized to perform Study 1 or Study 2 (n = 8, per study). Subjects received a primed, constant infusion of L-[ring-13C6]phenylalanine coupled with muscle biopsies of the vastus lateralis to measure rates of myofibrillar protein synthesis (MPS). Subjects in Study 2 were fed 25 g of whey protein immediately after an acute bout of unilateral resistance exercise.ResultsThere was no difference (P = 0.3) in rates of MPS determined using the steady-state precursor-product equation and determination of tracer incorporation between sequential biopsies 150 min apart or using plasma protein as the baseline enrichment, provided the infusion length was sufficient (230 ± 0.3 min). We also found that adding a modest amount of tracer (4% enriched), calculated based on the measured phenylalanine content of the protein (3.5%) in the drink, did not compromise steady-state conditions (slope of the enrichment curve not different from zero) in the plasma free or, more importantly, the IC pool (both P > 0.05).ConclusionsThese data demonstrate that the single biopsy approach yields comparable rates of muscle protein synthesis, provided a longer incorporation time is utilized, to that seen with a traditional two biopsy approach. In addition, we demonstrate that enriching protein-containing drinks with tracer does not disturb isotopic steady-state and thus both are reliable techniques to determine rates of MPS in humans.

Stable isotope tracing of fertilizer sulphur uptake by lodgepole pine: foliar responses

A plot-scale fertilizer sulphur (S) stable isotope tracer study was established in 2001 in the Sub-Boreal Spruce biogeoclimatic zone in central interior British Columbia where S deficiencies are common in lodgepole pine ( Pinus contorta var. latifolia Engelm. ex S. Wats.) stands. Treatments used operationally realistic applications of 300 kg N·ha–1 as urea and 100 kg S·ha–1 as either sulphate (SO4) or elemental S (S0). δ34S values of fertilizer S differed by &gt;9‰ from pretreatment δ34S values of total S in foliage at the two study sites (5.2‰ and 8.2‰). These differences enabled quantification of fertilizer uptake using isotopic analysis of post-treatment foliar S. Addition of K2SO4 with δ34S of +17.5‰ increased foliar δ34S by 3.5‰ and 6.6‰ at the two sites, respectively, in the year after treatment, indicating fertilizer contributions &gt;40% to foliar total S. For a S0 fertilizer application with a δ34S value of +19.3‰, foliar δ34S increases were smaller but steadily increased, resulting in an average tracer S uptake of ~20% over three years. These results confirmed the more rapid availability of S from SO4-based fertilizers and demonstrated the feasibility of field tracer experiments using stable S isotopes at natural abundance levels.

Nutrient removal from on-site domestic wastewater in horizontal subsurface flow reed beds in Ireland

In order to investigate the nutrient (namely nitrogen and phosphorus) removal efficiency and the governing internal dynamics of the most widely used wetland type, the horizontal subsurface flow reed bed, in receiving domestic septic tank and secondary effluent in a temperate climate such as Ireland, two systems were designed, constructed and rigorously monitored for a period of over 2 years. Nitrogen removal, as expected, was found to be poor across both reed beds, with only 29% removal of TN across the secondary treatment bed and 41% removal across the tertiary treatment bed, with little distinctive seasonal change. A 15N stable isotope tracer study revealed, in line with the results from the chemical analysis, that nitrogen kinetics in the secondary treatment bed were dominated by continuous plant litter decomposition and mineralisation processes converting stored org-N to NH 4-N indefinitely. Similar analysis on the tertiary treatment bed indicated that only limited denitrification of the oxidized forms of N was occurring in the anoxic environment of the bed, while NH 4-N and org-N were merely changing form on a cyclic basis. Removal of PO 4-P from the secondary and tertiary treatment beds was equally poor at rates of 45% and 22%, respectively. While at their maximum growth in the third year of operation, the total phosphorus in the stems and roots of the Phragmites australis in the secondary treatment bed equated to only 10% of the total P removed over the duration of the bed's operation. In the tertiary treatment bed, more seasonal variability was recorded with intermittent negative removal found during winter periods. This was somewhat more reflected in the P-uptake study for this bed with the roots and stems of the Typha and Iris containing phosphorus, which accounted for 31% of the overall mass removed.

Genetic determinants of apolipoprotein B-100 kinetics

We review stable isotope tracer studies of apolipoprotein B-100 (apoB) kinetics concerning genetic polymorphisms and mutations that affect human lipoprotein metabolism. In obese men, the allelic combination of the apoB signal peptide, SP24, and cholesteryl ester transfer protein, CETP B1B1, is independently associated with lower VLDL apoB secretion. Microsomal triglyceride transfer protein -493G/T carriers have reduced IDL apoB and LDL apoB production as compared with controls. Mutations in cholesterol transporters (ATP-binding cassette transporter G8 and Niemann-Pick C1 Like 1) are associated with reduced VLDL apoB secretion and increased LDL apoB production and catabolism. The ATP-binding cassette transporter G8 400K variant is a significant, independent predictor of VLDL apoB secretion. Mutations in lipases (lipoprotein lipase and hepatic lipase) and transfer proteins (lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein) alter their functional activity, which impact on VLDL and LDL kinetics. Mutations in genes that regulate intrahepatic apoB assembly and lipid substrate availability to the liver impact on VLDL apoB secretion. Lipoprotein tracer studies can provide functional insight into the potential impact of genetic polymorphisms in regulating apoB metabolism in humans.

The fate of cyanobacterial blooms in vegetated and unvegetated sediments of a shallow eutrophic lake: A stable isotope tracer study

An experiment using nitrogen stable isotope tracer ( 15N) was conducted to track the fate of nitrogen derived from cyanobacterial blooms and the effectiveness with which the seasonal blooms are retained by vegetated and unvegetated sediment in a large shallow eutrophic lake (Lake Taihu, China). 15N enriched Microcystis was injected into both unvegetated sediment and sediment occupied by common reed ( Phragmites australis) in the littoral zone. Nutrient retention by the vegetated sediment was greater than by the unvegetated sediment, resulting in higher δ 15N in the sediment nitrogen pool. The labeled Microcystis material was also distributed deeper into the vegetated sediment than the unvegetated sediment. A portion of the Microcystis-derived nitrogen was quickly assimilated, appearing first in the belowground biomass and subsequently in the aboveground biomass of the reed plants. The labeled nitrogen was found to support new growth as evidenced by 15N enrichment of new leaves. This study indicates that common reed beds in the littoral zone may play an important role in retention of sedimented planktonic materials.

The Effects of Recombinant Human Leptin on Visceral Fat, Dyslipidemia, and Insulin Resistance in Patients with Human Immunodeficiency Virus-Associated Lipoatrophy and Hypoleptinemia

Leptin deficiency is associated with dyslipidemia and insulin resistance in animals and humans with lipoatrophy; leptin replacement ameliorates these abnormalities. The objective of the study was to evaluate the effects of leptin therapy in lipoatrophic HIV-infected patients with dyslipidemia and hypoleptinemia. This was a 6-month, open-label, proof-of-principle pilot study. Metabolic ward studies were performed before and 3 and 6 months after leptin treatment. Participants included eight HIV-infected men with lipoatrophy, fasting triglycerides greater than 300 mg/dl, and serum leptin less than 3 ng/ml. Recombinant human leptin was given by sc injection (0.01 mg/kg and 0.03 mg/kg twice daily for successive 3 month periods). Measures included fat distribution by magnetic resonance imaging and dual-energy X-ray absorptiometry; fasting lipids; insulin sensitivity by euglycemic hyperinsulinemic clamp; endogenous glucose production, gluconeogenesis, glycogenolysis, and whole-body lipolysis by stable isotope tracer studies; oral glucose tolerance testing; liver fat by proton magnetic resonance spectroscopy; and safety. Visceral fat decreased by 32% (P = 0.001) with no changes in peripheral fat. There were significant decreases in fasting total (15%, P = 0.012), direct low-density lipoprotein (20%, P = 0.002), and non-high-density lipoprotein (19%, P = 0.005) cholesterol. High-density lipoprotein cholesterol increased. Triglycerides, whole-body lipolysis, and free fatty acids decreased during fasting and hyperinsulinemia. Fasting insulin decreased. Endogenous glucose production decreased during fasting and hyperinsulinemia, providing evidence of improved hepatic insulin sensitivity. Leptin was well tolerated but decreased lean mass. Leptin treatment was associated with marked improvement in dyslipidemia. Hepatic insulin sensitivity improved and lipolysis decreased. Visceral fat decreased with no exacerbation of peripheral lipoatrophy. Results from this pilot study suggest that leptin warrants further study in patients with HIV-associated lipoatrophy.

How relevant is the ratio of dietary n-6 to n-3 polyunsaturated fatty acids to cardiovascular disease risk? Evidence from the OPTILIP study

There has been much debate over the practical utility of the dietary ratio of n-6 to n-3 polyunsaturated fatty acids in optimizing the benefits of n-3 fatty acids (C18-C22) on cardiovascular health. This review examines the supporting evidence from the OPTILIP study within the context of the emerging consensus on the value of this dietary metric. The question of whether the ratio of n-6/n-3 polyunsaturated fatty acids or total amounts of dietary polyunsaturated fatty acids is of more importance to cardiovascular health has been addressed recently in a randomly controlled trial (OPTILIP) and in a stable isotope tracer study. These two studies were independently unanimous in concluding that the ratio of n-6/n-3 polyunsaturated fatty acids is of no value in modifying cardiovascular disease risk. The latter study also showed that the absolute amounts of dietary linoleic acid and alpha-linolenic acid are of relevance to the efficiency of conversion of alpha-linolenic acid to eicosapentaenoic acid and docosahexaenoic acid. This review should help to settle any outstanding controversy over the dietary ratio of n-6/n-3 polyunsaturated fatty acids. It reinforces current recommendations to increase the consumption of preformed eicosapentaenoic acid/docosahexaenoic acid in fish, and supports dietary measures to increase and decrease intakes alpha-linolenic acid and linoleic acid, respectively, to promote the endogenous synthesis of these longer chain n-3 polyunsaturated fatty acids.

Metabolism of α-linolenic acid in humans

α-Linolenic acid (18:3n-3) is essential in the human diet, probably because it is the substrate for the synthesis of longer-chain, more unsaturated n-3 fatty acids eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) which are required for tissue function. This article reviews the recent literature on 18:3n-3 metabolism in humans, including fatty acid β-oxidation, recycling of carbon by fatty acid synthesis de novo and conversion to longer-chain polyunsaturated fatty acids (PUFA). In men, stable isotope tracer studies and studies in which volunteers increased their consumption of 18:3n-3 show conversion to 20:5n-3 and 22:5n-3, but limited conversion to 22:6n-3. However, conversion to 18:3n-3 to 20:5n-3 and 22:6n-3 is greater in women compared to men, due possibly to a regulatory effect of oestrogen, while partitioning of 18:3n-3 towards β-oxidation and carbon recycling was lower than in men. These gender differences may be an important consideration in making dietary recommendations for n-3 PUFA intake.

Regulation of Folate-mediated One-carbon Metabolism by 10-Formyltetrahydrofolate Dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.

Assessing the effects of LXR agonists on cellular cholesterol handling: a stable isotope tracer study

The liver X receptors (LXRs) alpha and beta are responsible for the transcriptional regulation of a number of genes involved in cholesterol efflux from cells and therefore may be molecular targets for the treatment of cardiovascular disease. However, the effects of LXR ligands on cholesterol turnover in cells has not been examined comprehensively. In this study, cellular cholesterol handling (e.g., synthesis, catabolism, influx, and efflux) was examined using a stable isotope labeling study and a two-compartment modeling scheme. In HepG2 cells, the incorporation of 13C into cholesterol from [1-13C]acetate was analyzed by mass isotopomer distribution analysis in conjunction with nonsteady state, multicompartment kinetic analysis to calculate the cholesterol fluxes. Incubation with synthetic, nonsteroidal LXR agonists (GW3965, T0901317, and SB742881) increased cholesterol synthesis (approximately 10-fold), decreased cellular cholesterol influx (71-82%), and increased cellular cholesterol efflux (1.7- to 1.9-fold) by 96 h. As a consequence of these altered cholesterol fluxes, cellular cholesterol decreased (36-39%) by 96 h. The increased cellular cholesterol turnover was associated with increased expression of the LXR-activated genes ABCA1, ABCG1, FAS, and sterol-regulatory element binding protein 1c. In summary, the mathematical model presented allows time-dependent calculations of cellular cholesterol fluxes. These data demonstrate that all of the cellular cholesterol fluxes were altered by LXR activation and that the increase in cholesterol synthesis did not compensate for the increased cellular cholesterol efflux, resulting in a net cellular cholesterol loss.

Conversion of α-linolenic acid to longer-chain polyunsaturated fatty acids in human adults

The principal biological role of alpha-linolenic acid (alphaLNA; 18:3n-3) appears to be as a precursor for the synthesis of longer chain n-3 polyunsaturated fatty acids (PUFA). Increasing alphaLNA intake for a period of weeks to months results in an increase in the proportion of eicosapentaenoic acid (EPA; 20:5n-3) in plasma lipids, in erythrocytes, leukocytes, platelets and in breast milk but there is no increase in docosahexaenoic acid (DHA; 22:6n-3), which may even decline in some pools at high alphaLNA intakes. Stable isotope tracer studies indicate that conversion of alphaLNA to EPA occurs but is limited in men and that further transformation to DHA is very low. The fractional conversion of alphaLNA to the longer chain n-3 PUFA is greater in women which may be due to a regulatory effect of oestrogen. A lower proportion of alphaLNA is used for beta-oxidation in women compared with men. Overall, alphaLNA appears to be a limited source of longer chain n-3 PUFA in humans. Thus, adequate intakes of preformed long chain n-3 PUFA, in particular DHA, may be important for maintaining optimal tissue function. Capacity to up-regulate alphaLNA conversion in women may be important for meeting the demands of the fetus and neonate for DHA.