Amanda E. Jones

Eicosapentaenoic and docosapentaenoic acids are the principal products of α-linolenic acid metabolism in young men

The capacity for conversion of alpha-linolenic acid (ALNA) to n-3 long-chain polyunsaturated fatty acids was investigated in young men. Emulsified [U-13C]ALNA was administered orally with a mixed meal to six subjects consuming their habitual diet. Approximately 33 % of administered [13C]ALNA was recovered as 13CO2 on breath over the first 24 h. [13C]ALNA was mobilised from enterocytes primarily as chylomicron triacylglycerol (TAG), while [13C]ALNA incorporation into plasma phosphatidylcholine (PC) occurred later, probably by the liver. The time scale of conversion of [13C]ALNA to eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) suggested that the liver was the principal site of ALNA desaturation and elongation, although there was some indication of EPA and DPA synthesis by enterocytes. [13C]EPA and [13C]DPA concentrations were greater in plasma PC than TAG, and were present in the circulation for up to 7 and 14 d, respectively. There was no apparent 13C enrichment of docosahexaenoic acid (DHA) in plasma PC, TAG or non-esterified fatty acids at any time point measured up to 21 d. This pattern of 13C n-3 fatty acid labelling suggests inhibition or restriction of DHA synthesis downstream of DPA. [13C]ALNA, [13C]EPA and [13C]DPA were incorporated into erythrocyte PC, but not phosphatidylethanolamine, suggesting uptake of intact plasma PC molecules from lipoproteins into erythrocyte membranes. Since the capacity of adult males to convert ALNA to DHA was either very low or absent, uptake of pre-formed DHA from the diet may be critical for maintaining adequate membrane DHA concentrations in these individuals.

A method for separation of phosphatidylcholine, triacylglycerol, non-esterified fatty acids and cholesterol esters from plasma by solid-phase extraction.

Efficient isolation of individual lipid classes is a critical step in the analysis of plasma and lipoprotein fatty acid compositions. Whilst good separations of total lipid extracts are possible by TLC, this method is time consuming and a major rate-limiting step when processing large numbers of specimens. A method for rapid separation of phosphatidylcholine (PC), non-esterified fatty acids (NEFA), cholesterol ester (CE) and triacylglycerol (TAG) from total plasma lipid extracts by solid-phase extraction (SPE) using aminopropyl silica columns has been developed and validated. Following initial separation of polar and neutral lipids, individual classes were isolated by application of solvents with increasing polarity. Recoveries for combined plasma extraction with chloroform-methanol and SPE were (%): PC 74.2 (SD 7.5), NEFA 73.6 (SD 8.3), CE 84.9 (SD 4.9), and TAG 86.8 (SD 4.9), which were significantly greater for TAG and NEFA than by TLC Both GC-flame ionisation detector and GC-MS analysis of fatty acid methyl esters demonstrated that there was no cross-contamination between lipid classes. Measurements of repeatability of fatty acid composition for TAG, PC, CE and NEFA fractions showed similar CV for each fatty acid. The magnitude of the CV appeared to be related inversely to the fractional fatty acid concentration, and was greatest at concentrations of less than 1 g/100 g total fatty acids. There was no evidence of selective elution of individual fatty acid or CE species. In conclusion, this method represents an efficient, rapid alternative to TLC for isolation of these lipid classes from plasma.

Chylomicron particle size and number, factor VII activation and dietary monounsaturated fatty acids.

This study evaluated the effects of substituting dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) on postprandial chylomicron (triacylglycerol (TAG), apolipoprotein B-48 (apo B-48) and retinyl ester (RE)), chylomicron particle size and factor VII (FVII) response when subjects were given a standard meal. In a controlled sequential design, 51 healthy young subjects followed an SFA-rich diet (Reference diet) for 8 weeks after which half of the subjects followed a moderate MUFA diet (n=25) and half followed a high MUFA diet (n=26) for 16 weeks. Fasting lipoprotein and lipid measurements were evaluated at baseline and at 8-week intervals during the Reference and MUFA diets. In 25 of the subjects (n=12 moderate MUFA, n=13 high MUFA), postprandial responses to a standard test meal containing RE and 13C-tripalmitin were investigated at the end of the Reference and the MUFA diet periods. Although there were no differences in the postprandial lipid markers (TAG, RE, 13C-TAG) on the two diets, the postprandial apo B-48 response (incremental area under the curve (IAUC)) was reduced by 21% on the moderate MUFA diet (NS) and by 54% on the high MUFA diet (P<0.01). The postprandial peak concentrations of apo B-48 were reduced by 33% on the moderate MUFA diet (P<0.01) and 48% on the high MUFA diet (P<0.001). Fasting values for factor VII activity (FVIIc), activated factor VII (FVIIa) or factor VII antigen (FVIIag) did not differ significantly when subjects were transferred from Reference to MUFA diets. However, the postprandial increases in coagulation FVII activity (FVIIc) were 18% lower and of activated FVII (FVIIa) were 17% lower on the moderate MUFA diet (NS). Postprandial increases in FVIIc and FVIIa were 50% (P<0.05) and 29% (P<0.07) lower on the high MUFA diet and the area under the postprandial FVIIc response curve (AUC) was also lower on the high MUFA diet (P<0.05). Significantly higher ratios of RE:apo B-48 (P<0.001) and 13C-palmitic acid:apo B-48 (P<0.01) during both MUFA diets suggest that the CMs formed carry larger amounts of dietary lipids per particle, reflecting an adaptation to form larger lipid droplets in the enterocyte when increased amounts of dietary MUFAs are fed. Smaller numbers of larger chylomicrons may explain attenuated activation of factor VII during the postprandial state when the background diet is rich in MUFA.

Effect of fatty acid chain length and saturation on the gastrointestinal handling and metabolic disposal of dietary fatty acids in women.

The gastrointestinal handling and metabolic disposal of [1-13C]palmitic acid, [1-13C]stearic acid and [1-13C]oleic acid administered within a lipid-casein-glucose-sucrose emulsion were examined in normal healthy women by determining both the amount and nature of the 13C label in stool and label excreted on breath as 13CO2. The greatest excretion of 13C label in stool was in the stearic acid trial (9.2% of administered dose) whilst comparatively little label was observed in stool in either the palmitic acid (1.2% of administered dose) or oleic acid (1.9% of administered dose) trials. In both the palmitic acid and oleic acid trials, all of the label in stool was identified as being present in the form in which it was administered (i.e. [13C]palmitic acid in the palmitic acid trial and [13C]oleic acid in the oleic acid trial). In contrast, only 87% of the label in the stool in the stearic acid trial was identified as [13C]stearic acid, the remainder was identified as [13C]palmitic acid which may reflect chain shortening of [1-13C]stearic acid within the gastrointestinal tract. Small, but statistically significant, differences were observed in the time course of recovery of 13C label on breath over the initial 9 h of the study period (oleic acid = palmitic acid > stearic acid). However, when calculated over the 24 h study period, the recovery of the label as 13CO2 was similar in all three trials (approximately 25% of absorbed dose). These results support the view that chain length and degree of unsaturation may influence the gastrointestinal handling and immediate metabolic disposal of these fatty acids even when presented within an emulsion.

The gastrointestinal handling and metabolism of [1-13C]palmitic acid in healthy women

The gastrointestinal handling and metabolism of [1-13C]palmitic acid given as the free fatty acid was examined in six healthy women by measuring the excretion of13C-label in stool and in breath as13CO2. The gastrointestinal handling of [1-13C]palmitic acid was compared with the apparent absorption of dietary lipid by measuring lipid losses in stool. The variation both within and between subjects was determined by repeating the study in the same individuals on separate occasions. The time course for excretion of label in stool over the five-day study period followed a common pattern, with most of the label excreted over the first two days of the stool collection.13C-Label excreted in stool over the five-day study period was 14.3±9.8% of that administered and on repeating the trial was 31.6±24.7% (not significantly different due to variability); there was poor agreement within subjects. Lipid excreted in stool expressed as a percentage of ingested lipid was 5.2±4.4% in Trial 1 and 5.9±4.0% in Trial 2, and was the same in each individual on repeating the trial. There was no clear relationship between the excretion of13C-label and lipid in stool (Trial 1:R=−0.43,P>0.40; Trial 2:R=−0.02,P>0.97). On the first occasion, 22.0±4.5% of the administered label was excreted on breath over the 15-h study period and on repeating the trial was 15.8±9.5% (not significantly different) with poor repeatability in a given individual. There was an inverse relationship between the proportion of13C-label excreted in stool and that excreted on breath in Trial 1 (R=−0.80,P>0.06) with a weaker association observed in Trial 2 (R=−0.49,P>0.32). Correcting for differences in the apparent absorption of label reduced the variability in its excretion in breath observed between subjects, particularly in Trial 2. It is concluded that although there are differences in the gastrointestinal handling of [1-13C]palmitic acid both within and between healthy adults, the postprandial oxidation of absorbed substrate was similar. The assumptions underlying these observations need to be examined by characterizing the nature of13C-label in stool.

Effect of meal sequence on postprandial lipid, glucose and insulin responses in young men

Objective: To investigate whether the postprandial changes in plasma triacylglycerol (TAG), nonesterified fatty acids (NEFA), glucose and insulin concentrations in young men were the same if an identical meal was fed at breakfast and lunch, and if the response to lunch was modified by consumption of breakfast.Methods: In two trials (1 and 2) healthy subjects (age 22±1 y, body mass index 22±2 kg/m2) were fed the same mixed macronutrient meal at breakfast at 08:00 h and lunch at 14:00 h. In the third trial, no breakfast was fed and the overnight fast extended until lunch at 14:00 h. Addition of [1,1,1-13C]tripalmitin to one meal in each trial was used to distinguish between endogenous and meal-derived lipids.Results: The postprandial changes in TAG, NEFA and glucose concentrations were similar in trials 1 and 2. The change in plasma total TAG concentration was about two fold less (P<0.05) after lunch compared to breakfast. Postprandial NEFA suppression was the same after breakfast and lunch. Glucose and insulin responses were significantly greater following lunch suggesting decreasing insulin sensitivity during the day. Consumption of breakfast did not alter the postprandial total TAG or NEFA responses after lunch. Measurement of [13C]palmitic acid concentration showed that handling of TAG and NEFA from the meal was the same after breakfast and lunch, and was not altered by consumption of breakfast.Conclusions: Overall, these data suggest that in young, healthy men regulation of plasma TAG from endogenous sources, principally VLDL, but not chylomicrons during the postprandial period leads to differences in the magnitude of lipaemic response when the same meal was consumed at breakfast or at lunch 6 h later.

The effect of age and gender on the metabolic disposal of [1-13C]palmitic acid

Objective: To examine the effect of age and gender on the metabolic disposal of [1-13C] palmitic acid.Design: Cross-sectional.Setting: Clinical Nutrition and Metabolism Unit at Southampton General Hospital, Institute of Human Nutrition, University of Southampton.Subjects and measurements: Twelve children (5 boys and 7 girls; aged 5–10 y) and six men (BMI 23.3±2.6 kg/m2; aged 20–30 y) were recruited. Following oral administration of a bolus dose of [1-13C]palmitic acid (10 mg/kg body weight) consumed with a test meal (1667 kJ) the excretion of 13C-label was measured on breath as 13CO2 over 24 h and in stool over 5 d to account for differences in absorption of [1-13C]palmitic acid. The 13C-enrichment of samples was determined by continuous flow-isotope ratio mass spectrometry. Net substrate oxidation was estimated from gaseous exchange measurements in the postabsorptive state and over 6 h postprandially.Results: The excretion of 13CO2 on breath varied between subjects both in the pattern and amount excreted over 24 h. Breath 13CO2 was not different between boys (61.0±22.4% of absorbed dose) and girls (54.2±17.9% of absorbed dose). The excretion of breath 13CO2 was less in the men (35.1±9.3% of absorbed dose; P=0.005) and that observed previously by our group in women (30.7±6.7% of absorbed dose; P=0.005) than in the children. Net fat oxidation was greater in the children in both the postabsorptive (2.43±0.78 g/h) and postprandial (11.89±3.13 g/6 h) states than in the men (0.93 g/h±1.50; P=0.016; 9.86±10.53 g/6 h; NS) and women studied previously (0.53±0.68 g/h; P=0.003; 0.03±3.21 g/6 h; P=0.001).Conclusions: Our observations that children oxidised nearly twice the amount of [1-13C] palmitic acid than adults in conjunction with greater net fat oxidation in children than adults in both the postabsorptive and postprandial states should be considered before current UK dietary recommendations for fat and saturated fats, developed for adults, are applied to growing children. For dietary recommendations to be developed further more information is required, particularly in groups of infants and the elderly, about the factors that influence the postprandial handling of dietary fat.Sponsorships: This work was supported by The Ministry of Agriculture, Fisheries and Food, Scientific Hospital Supplies, UK Ltd. and The Wessex Medical Trust.

Gastrointestinal handling of [1-13C]palmitic acid in healthy controls and patients with cystic fibrosis

AIM To examine the gastrointestinal handling of [1-13C]palmitic acid given as the free acid by measuring the excretion of 13C label in stool in 16 healthy children and 11 patients with cystic fibrosis on their habitual enzyme replacement treatment. METHODS After an overnight fast, each child ingested 10 mg/kg body weight [1-13C]palmitic acid with a standardised test meal of low natural 13C abundance. A stool sample was collected before the test and all stools were collected thereafter for a period of up to five days. The total enrichment of 13C in stool and the species bearing the13C label was measured using isotope ratio mass spectrometry. RESULTS The proportion of administered13C label excreted in stool was 24.0% (range 10.7–64.9%) in healthy children and only 4.4% (range 1.2–11.6%) in cystic fibrosis patients. The enrichment of 13C in stool was primarily restricted to the species consumed by the subjects (that is as palmitic acid). CONCLUSION There does not appear to be a specific defect in the absorption of [1-13C]palmitic acid in patients with cystic fibrosis. The reasons why cystic fibrosis patients appear to absorb more of this saturated fatty acid than healthy children is not clear and requires further investigation.

Metabolic handling of 13C labelled tripalmitin in healthy controls and patients with cystic fibrosis

AIM To examine the gastrointestinal handling and metabolic disposal of emulsified [1-13C]palmitic acid esterified into a triglyceride in nine healthy children and seven patients with cystic fibrosis on enzyme replacement treatment. METHODS After an overnight fast, each child was given 10 mg/kg body weight [1,1,1-13C]tripalmitin with a standardised test meal of low natural 13C abundance. The total enrichment of13C was measured using isotope ratio mass spectrometry in stool collected for a period of up to five days and in breath samples collected over a 24 hour period. RESULTS The mean proportion of administered 13C label excreted in stool was 6% (range, 1–12.7%) in healthy children and 24.6% (range, 0–64%) in patients with cystic fibrosis. Healthy children excreted 31.3% of the administered label on their breath (range, 14.2–42.9%). Correcting the excretion of administered 13C label on the breath for differences in digestion and absorption in patients with cystic fibrosis increased the difference between individuals from 0–31.3% of administered dose (mean, 17.9%) to 0–49.1% of absorbed dose (mean, 23.2%) and was poorly related to the amount of13C label in stool. CONCLUSION Measurements of breath 13CO2 do not consistently reflect the gastrointestinal handling of emulsified 13C labelled tripalmitin because of differences in digestion and absorption in cystic fibrosis. Further studies need to examine whether “breath tests” alone can predict with confidence the gastrointestinal handling of other 13C labelled triglycerides and fatty acids.

Stable-isotope method for determining the gastrointestinal handling of [1-13C]palmitic acid

The 13C enrichment in individual fatty acids extracted from human feces following the oral administration of [1-13C]palmitic acid has been determined using a novel approach based upon gas chromatography-isotope ratio mass spectrometry. The method was established and tested for precision and repeatability. Analytical precision was determined from 10 repeated injections of a sample containing 16∶0 and 18∶0 with levels of δ13C abundance measured at −34.01±0.60 and −23.62±0.95 delta per mil (parts per thousand) (‰), respectively (mean±SD). For the repeatability study, measurement of enrichment of the same mixture of unlabeled fatty acid methyl ester (FAME) standards (13∶0, 14∶0, 16∶0, and 18∶0) was found to have standard deviations (0.45, 0.56, 1.46 and 1.54‰ respectively). When labeled [1-13C]palmitic acid was serially diluted with naturally enriched palmitic acid, a linear relationship was obtained to a dilution of 10% enriched compound (530‰). FAME were prepared from two fecal samples from a normal healthy adult; the first, a baseline specimen, containing no added label and the second, followed a single oral dose of [1-13C]palmitic acid and was enriched. Enrichment in 13C was confined to the solvent-soluble fraction following lipid extraction, and was only identified with prior acidification. The enrichments were measured in triplicate, baseline sample −32.66±0.5‰ enriched sample +268.61±8.0‰ Enrichment was restricted to the labeled species consumed, 16∶0. The methodology described here allows for the separation of compounds prior to the determination of enrichment and can be utilized to contribute to a more complete description of the gastrointestinal handling of labeled substrates than previously obtained.