A 13CO2 Enrichment Experiment to Study the Synthesis Pathways of Polyunsaturated Fatty Acids of the Haptophyte Tisochrysis lutea

The production of polyunsaturated fatty acids (PUFA) in Tisochrysis lutea was studied using the gradual incorporation of a 13C-enriched isotopic marker, 13CO2, for 24 h during the exponential growth of the algae. The 13C enrichment of eleven fatty acids was followed to understand the synthetic pathways the most likely to form the essential polyunsaturated fatty acids 20:5n-3 (EPA) and 22:6n-3 (DHA) in T. lutea. The fatty acids 16:0, 18:1n-9 + 18:3n-3, 18:2n-6, and 22:5n-6 were the most enriched in 13C. On the contrary, 18:4n-3 and 18:5n-3 were the least enriched in 13C after long chain polyunsaturated fatty acids such as 20:5n-3 or 22:5n-3. The algae appeared to use different routes in parallel to form its polyunsaturated fatty acids. The use of the PKS pathway was hypothesized for polyunsaturated fatty acids with n-6 configuration (such as 22:5n-6) but might also exist for n-3 PUFA (especially 20:5n-3). With regard to the conventional n-3 PUFA pathway, Δ6 desaturation of 18:3n-3 appeared to be the most limiting step for T. lutea, “stopping” at the synthesis of 18:4n-3 and 18:5n-3. These two fatty acids were hypothesized to not undergo any further reaction of elongation and desaturation after being formed and were therefore considered “end-products”. To circumvent this limiting synthetic route, Tisochrysis lutea seemed to have developed an alternative route via Δ8 desaturation to produce longer chain fatty acids such as 20:5n-3 and 22:5n-3. 22:6n-3 presented a lower enrichment and appeared to be produced by a combination of different pathways: the conventional n-3 PUFA pathway by desaturation of 22:5n-3, the alternative route of ω-3 desaturase using 22:5n-6 as precursor, and possibly the PKS pathway. In this study, PKS synthesis looked particularly effective for producing long chain polyunsaturated fatty acids. The rate of enrichment of these compounds hypothetically synthesized by PKS is remarkably fast, making undetectable the 13C incorporation into their precursors. Finally, we identified a protein cluster gathering PKS sequences of proteins that are hypothesized allowing n-3 PUFA synthesis.

Continuous Estimation of CO2 Production during Exercise

A new method was developed for continuous isotopic estimation of human whole body CO2 rate of appearance (Ra) during non-steady state exercise. The technique consisted of a breath-by-breath measurement of 13CO2 enrichment (E) and a real-time fuzzy logic feedback system which controlled NaH13CO3 infusion rate to achieve an isotopic steady state. Ra was estimated from the isotope infusion rate and body 13CO2 enrichment which was equal to E at the isotopic steady state. During a non-steady state incremental cycle exercise (5 w/min or 10 w/min), NaH13CO3 infusion rate was successfully increased by the action of feedback controller so as to keep E constant.

Metabolic availability of medium-chain triglycerides coingested with carbohydrates during prolonged exercise

The present study examined the metabolic response to medium-chain triglycerides (MCTs) ingestion with or without carbohydrates (CHOs). Eight well-trained athletes cycled 4 x 180 min at 50% maximal work rate (57% maximal O2 consumption). Subjects drank a bolus of 4 ml/kg at the start and 2 ml/kg every 20 min during exercise of either a 15% (214 g) CHO solution (CHO trial), an equicaloric 149 g CHO-29 g MCT suspension (CHO+MCT trial), 214 g CHO [high CHO (HCHO)]-29 g MCT suspension (HCHO+MCT trial) or 29 g MCT solution (MCT trial). Exogenous MCT oxidation was measured by adding a [1,1,1–13C]trioctanoate tracer to the MCT oil. 13CO2 enrichment of breath samples were measured every 15 min. During the second hour (60- to 120-min period), the amount of MCT oxidized was 72% of the amount ingested during the CHO+MCT trial, whereas during the MCT trial only 33% was oxidized. The rate of MCT oxidation increased more rapidly during the HCHO+MCT and CHO+MCT trials compared with the MCT trial, yet in all three cases the oxidation rate stabilized at 0.12 g/min during 120–180 min of exercise. It is concluded that more MCTs are oxidized when ingested in combination with CHOs. Data do confirm the hypothesis that oral MCTs might serve as an energy source in addition to glucose during exercise because the metabolic availability of MCTs was high during the last hour of exercise, with oxidation rates being approximately 70% of the ingestion rate.

Breath 13CO2 background enrichment during exercise: diet-related differences between Europe and America

A traditional North American diet contains a high percentage of carbohydrates (CHO) derived from C4 plants (maize, sugar cane), whereas a European diet contains primarily CHO derived from C3 plants (potato, sugar beet). The natural 13C enrichment of the first type of CHO is higher than that of the latter type. 13CO2 production from orally ingested C4 plant-derived CHO can, therefore, be used to quantify oxidation rates of orally ingested CHO at rest and during exercise. Recently it has been shown that oxidation rates assessed this way in North Americans should be corrected for an increase in breath background 13CO2 during exercise. We hypothesized that the indicated difference in metabolic origin of CHO would imply that no such correction is required for subjects on a European diet. We therefore studied changes from rest in breath 13CO2 enrichment in Dutch volunteers during cycle ergometry at 65% maximal work load (experiment 1, 2h, 6 subjects) and 70% maximal oxygen uptake (experiment 2, 90 min, 8 subjects) while ingesting water (experiments 1 and 2) and potato starch-derived glucose (experiment 2). Experiment 1 was done before and after careful instruction of the subjects to refrain from nutrient sources potentially containing CHO of C4 metabolic origin. No significant changes from rest 13CO2 enrichment were observed in tests with water and potato-derived glucose ingestion in subjects who excluded CHO of C4 metabolic origin from their diet.

Nitrogen sparing by 2-ketoisocaproate in parenterally fed rats

In rats receiving total parenteral nutrition with or without sodium 2-ketoisocaproate (KIC; 2.48 g.kg-1.day-1), L-[1-13C]leucine and [1–14C]KIC were constantly infused for 6 h. CO2 production, 14CO2 production, 13CO2 enrichment, urinary urea nitrogen (N) plus ammonia N and total urinary N were measured. Whole body protein synthesis (S) was calculated in non-KIC-infused rats and also in unfed rats infused with [1–14C]leucine from fractional oxidation of labeled leucine (1-F), where F is fractional utilization for protein synthesis, and urea N plus ammonia N excretion (C) as S = C x F/(1-F). Addition of KIC caused a significant reduction in N excretion and a significant improvement in N balance. Fractional oxidation of labeled KIC increased, whereas fractional utilization of labeled KIC for protein synthesis decreased, but the extent of incorporation of infused KIC into newly synthesized protein (as leucine) amounted to at least 40% of the total rate of leucine incorporation into newly synthesized whole body protein. We conclude that addition of KIC spares N in parenterally fed rats and becomes a major source of leucine for protein synthesis.

Effect of substrate intake and physiological state on background 13CO2 enrichment

The natural enrichment of 13C in energy substrates varies, and this variation must be taken into account when stable isotopic tracers are used in metabolic studies. This is conventionally accomplished by measuring background samples taken before the tracer infusion begins and subtracting these values from postinfusion values. Whereas this approach is satisfactory if no perturbation occurs between the collection of the background samples and the collection of postinfusion sample, the data presented in this paper show that any change in the metabolic state can significantly alter the background enrichment of expired CO2. This study not only confirmed that the introduction of natural energy sources may alter the background enrichment of CO2, but we also found that changes in substrate oxidation induced by different physiological states, such as exercise, can cause significant changes in expired CO2 enrichments. Conclusions from studies in which oxidation of substrates were measured by means of a 13C tracer but potential changes in background enrichments were not accounted for must, therefore, be reassessed.

Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-3C]leucine

Leucine metabolism in vivo can be determined from a primed, continuous infusion of L-[1-13C]leucine by measuring, at isotopic steady state, plasm [-13C]leucine enrichment, expired 13CO2 enrichment, and CO2 production rate. With an appropriate priming dose of L-[1-13C]leucine and NaH13CO3, isotopic steady state is reached in less than 2 h, and the infusion is completed in 4 h. The method can determine rates of leucine turnover, oxidation, and incorporation into protein with typical relative uncertainties of 2, 10, and 4%, respectively. The method requires no more than 1 ml of blood and uses stable isotope rather than radioisotope techniques. Thus, the method is applicable to studies of human beings of all ages. L-[1-13C]leucine may be infused with a second amino acid labeled with 15N for simultaneous determination of the kinetics of two amino acids.