Effect of 1- and 2-Month High-Dose Alpha-Linolenic Acid Treatment on 13 C-Labeled Alpha-Linolenic Acid Incorporation and Conversion in Healthy Subjects.

SCOPE: The study aims at identifying 1) the most sensitive compartment among plasma phospholipids, erythrocytes, and LDL for studying alpha-linolenic acid (ALA) conversion, and 2) whether ALA incorporation and conversion is saturable after administration of 13 C-labeled ALA-rich linseed oil (LO). The effect of a daily intake of 7 g nonlabeled LO (>43% w/w ALA) for 1 month after bolus administration of 7 g 13 C-labeled LO on day 1, and for 2 months after bolus administration of 7 g 13 C-labeled LO on day 1 and day 29 on 13 C-ALA incorporation and conversion into its higher homologs is investigated in healthy volunteers. METHODS AND RESULTS: Incorporation and conversion of LO-derived 13 C-labeled ALA is quantified by applying compartmental modeling. After bolus administration, a fractional conversion of approximately 30% from 13 C-ALA to 13 C-DHA is calculated as reflected by the LDL compartment. Treatment with LO for 8 weeks induces a mean reduction of 13 C-ALA conversion to 13 C-DHA by 48% as reflected by the LDL compartment, and a mean reduction of the 13 C-ALA incorporation into LDL by 46%. CONCLUSION: A 2-month dietary intake of a high dose of LO is sufficient to reach saturation of ALA incorporation into LDL particles, which are responsible for ALA distribution in the body.

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep.

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion.

Sources of hepatic glycogen synthesis following a milk-containing breakfast meal in healthy subjects.

During feeding, dietary galactose is a potential source of hepatic glycogen synthesis; but its contribution has not been measured to date. In the presence of deuterated water ((2)H(2)O), uridine diphosphate (UDP)-glucose derived from galactose is not enriched, whereas the remainder derived from glucose-6-phosphate (G6P) is enriched in position 2 to the same level as body water, assuming complete G6P-fructose-6-phosphate (F6P) exchange. Hence, the difference between UDP-glucose position 2 and body water enrichments reflects the contribution of galactose to glycogen synthesis relative to all other sources. In study 1, G6P-F6P exchange in 6 healthy subjects was quantified by supplementing a milk-containing breakfast meal with 10 g of [U-(2)H(7)]glucose and quantifying the depletion of position 2 enrichment in urinary menthol glucuronide. In study 2, another 6 subjects ingested (2)H(2)O and acetaminophen followed by an identical breakfast meal with 10 g of [1-(13)C]glucose to resolve direct/indirect pathways and galactose contributions to glycogen synthesis. Metabolite enrichments were determined by (2)H and (13)C nuclear magnetic resonance. In study 1, G6P-F6P exchange approached completion; therefore, the difference between position 2 and body water enrichments in study 2 (0.20% ± 0.03% vs 0.27% ± 0.03%, P < .005) was attributed to galactose glycogenesis. Dietary galactose contributed 19% ± 3% to glycogen synthesis. Of the remainder, 58% ± 5% was derived from the direct pathway and 22% ± 4% via the indirect pathway. The contribution of galactose to hepatic glycogen synthesis was resolved from that of direct and indirect pathways using a combination of (2)H(2)O and [1-(13)C]glucose tracers.

A mass balance approach to identify and compare differential routing of 13C-labeled carbohydrates, lipids, and proteins in vivo.

All animals route assimilated nutrients to their tissues where they are used to support growth or are oxidized for energy. These nutrients are probably not allocated homogeneously among the various tissue and are more likely to be preferentially routed toward some tissues and away from others. Here we introduce an approach that allows researchers to identify and compare nutrient routing among different organs and tissues. We tested this approach by examining nutrient routing in birds. House sparrows Passer domesticus were fed a meal supplemented with one of seven (13)C-labeled metabolic tracers representing three major classes of macronutrients, namely, carbohydrates, amino acids, and fatty acids. While these birds became postabsorptive (2 h after feeding), we quantified the isotopic enrichment of the lean and lipid fractions of several organs and tissues. We then compared the actual (13)C enrichment of various tissue fractions with the predictions of our model to identify instances where nutrients were differentially routed and found that different classes of macronutrients are uniquely routed throughout the body. Recently ingested amino acids were preferentially routed to the lean fraction of the liver, whereas exogenous carbohydrates were routed to the brain and the lipid fraction of the liver. Fatty acids were definitively routed to the heart and the liver, although high levels of palmitic acid were also recovered in the adipose tissue. Tracers belonging to the same class of molecules were not always routed identically, illustrating how this technique is also suited to examine differences in nonoxidative fates of closely related molecules. Overall, this general approach allows researchers to test heretofore unexamined predictions about how animals allocate the nutrients they ingest.

No association between serotonin 5-HT 1A receptors and spirituality among patients with major depressive disorders or healthy volunteers.

An earlier study (Borg et al., Am J Psychiatry 2003) found an inverse correlation between [carbonyl-(11)C]WAY-100635 ligand binding to 5-HT(1A) receptors and scores for self-transcendence, but no other of the six dimensions of the Temperament and Character Inventory, in a group of healthy males. The aim of this study was to investigate if the finding of an inverse correlation between spirituality and 5-HT(1A) could be seen in patients suffering from major depressive disorder or replicated among healthy volunteers. A total of 23 patients with major depressive disorder and 20 healthy volunteers were examined with PET using [carbonyl-(11)C]WAY-100635 as the radioligand. The personality traits were measured using the Finnish version of the Temperament and Character Inventory and correlated with ligand binding (BP). No significant correlations were found between the different Temperament and Character Inventory subscales and BP in any of the studied brain regions (amygdala, anterior cingulate cortex, dorsal raphe nuclei, dorsolateral prefrontal cortex, angular gyrus, inferior, middle, and superior temporal gyri, medial prefrontal cortex orbitofrontal cortex, hippocampus, insular cortex, subgenual anterior cingulate cortex, supramarginal gyrus, ventrolateral prefrontal cortex, and posterior cingulate cortex). These results do not support the idea that the serotonin system forms the biological basis of spiritual experiences among patients suffering from major depressive disorder or among healthy volunteers.

Alfentanil increases cortical dopamine D2/D3 receptor binding in healthy subjects.

Animal studies have shown that opioids modulate the function of dopaminergic neurons. The effect of alfentanil on cortical and thalamic binding of the D2/D3 receptor ligand [(11)C]FLB 457 was evaluated in eight healthy subjects with positron emission tomography. The simplified reference tissue model was used to calculate tracer binding potential (BP) during a baseline condition and target-controlled infusion of alfentanil, and the results were analyzed using a comparison group not receiving opioid. Behavioral and analgesic effects of alfentanil were also evaluated. In the region-of-interest analysis, alfentanil increased the BP of [(11)C]FLB 457 in the medial frontal cortex (P=0.0027), dorsolateral prefrontal cortex (P=0.027) superior temporal cortex (P=0.028), and medial thalamus (P=0.003) These results were confirmed in a voxel-based analysis, which further revealed an opioid-induced increase in [(11)C]FLB 457 BP in the anterior cingulate cortex (P<0.001). Alfentanil induced euphoria (P=0.003) and analgesia (P=0.006) Cheerfulness (r=0.918, P=0.001) and euphoria (r=0.982, P<0.001) were associated with increased BP of [(11)C]FLB 457 in the left posterior cingulate cortex, but the analgesic effect of alfentanil did not correlate with changes in [(11)C]FLB 457 BP. The results of this study demonstrate opioid-dopamine interactions in frontal and temporal cortical regions and the thalamus in healthy subjects. Increased D2/D3 tracer binding during opioid infusion may reflect decreased synaptic dopamine levels. The association of the uplifting effect of alfentanil with increased D2/D3 binding in the posterior cingulate cortex suggests that cortical dopamine may be involved in the behavioral effects of opioids.