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Stable isotope tracing and triglyceride kinetics
ARTICLE IN PRESS Clinical Nutrition (2004) 23, 439–440
www.elsevier.com/locate/clnu
EDITORIAL
Stable isotope tracing and triglyceride kinetics Triglycerides synthesized in the liver are secreted into plasma as a part of very-low density lipoproteins (VLDL). These particles permit the delivery of lipid fuel from the liver to other tissues for energy metabolism. Understanding VLDL-triglyceride kinetics in vivo, in humans, has important physiological and clinical implications. This kind of information will be essential to an understanding of the regulation of plasma lipid transport in the fasting and postprandial states and of the subtle changes that occur in those at risk for coronary heart disease. The paradigm of dyslipidemia in the insulin resistance syndrome associated with visceral obesity as well as type 2 diabetes typically consists of increased plasma levels and flux of VLDL-triglycerides from the liver. A variety of approaches have been used to determine in vivo, in humans, endogenous triglyceride synthesis, each with certain limitations. The direct splanchnic release of triglyceride in VLDL has been determined in humans by measuring the arteriovenous difference in triglyceride concentrations across the splanchnic tissues in combination with splanchnic blood flow.1 This invasive approach provides the most unambiguous information, but precision is low because of the technical problems related to accurate determinations of concentrations and blood flow. The use of isotopic tracers for investigating lipid metabolism in human subjects has increased substantially over the last decade due to marked improvements in analytical technologies and better availability of suitable tracers. The most common isotopic approaches to quantify VLDLtriglyceride kinetics are based on the fact that labelled fatty acids (e.g., palmitate) and glycerol infused into the circulation are readily taken up by the liver to be incorporated into newly synthesized triglycerides. These tracers can be administered as a constant infusion or a bolus injection to determine the monoexponential slopes of the increase2 or of the decline3 in plasma labelled VLDLtriglyceride enrichments, respectively. Compartimental modelling may improve the accuracy of determining VLDL-triglyceride kinetics by account-
ing for tracer recycling.2–4 Recently, the precursor– product approach to determine triglyceride synthesis has been improved by calculating intrahepatic precursor enrichments based on mass isotopomer distribution analysis.2,5 The experiments to determine endogenous triglyceride synthesis based on tracer incorporation are non-invasive and easy to carry out. Nonetheless, the relatively complex mathematical models to derive the kinetic parameters of interest and the uncertainties about the number and location of interacting compartments have made it impossible to obtain reliable and precise quantification of triglyceride kinetics to date. Beside the use of the precursor–product relationship, isotopic tracers can be employed in vivo, in humans, according to the tracer dilution technique. This is a suitable approach to determine appearance in a defined compartment of turning over substrates such as glucose from gluconeogenesis and glycogenolysis, amino acids from protein degradation or glycerol and free fatty acids from triglyceride hydrolysis. This method is conceptually straightforward and does not rely on complicated models and unverifiable assumptions. It requires steady state condition that is easily demonstrable by serial determinations of isotopic enrichments in the accessible compartments. This methods, at least in theory, is also suitable for determination of appearance rates in plasma of large and complex molecules such as proteins and lipoprotein-bound lipids. Nonetheless, a major limitation relies on the fact that pre-labelling of large and complex molecules is technically difficult and involves the risk of intravenous infusion of impure preparations in humans. In this issue of Clinical Nutrition, Sidossis et al. described a novel approach to quantify VLDL-triglyceride production rates in humans according to the traditional isotope dilution technique.6 The method involved the invivo labelling of the glycerol backbone of VLDLtriglyceride with [U-13C3]glycerol, plasmapheresis, isolation of the endogenously labelled VLDL, and autologous reinfusion on a separate occasion. The labelled VLDL-triglycerides were synthesized in vivo
0261-5614/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.clnu.2004.06.002
ARTICLE IN PRESS 440
few days prior to the infusion studies. The authors demonstrated a clear isotopic steady state during the autologous reinfusion of labelled VLDL in both the postabsorptive and fed states. This allowed the use of simple tracer dilution equations for kinetic calculations, thereby avoiding assumptions on the heterogeneity of endogenous VLDL pools. The calculated rate of triglyceride appearance was in good agreement with that obtained by the arteriovenous triglyceride balance across the splanchnic bed.1 Despite the clear advantages of this straightforward approach, the re-injection of biologically labelled plasma VLDL has disadvantages in terms of invasivity and logistics and may be impractical for large scale use. An alternative to the in-vivo prelabelling of VLDL-triglycerides with isotopic tracers of glycerol is the in-vitro iodination of VLDLapoproteins. This procedure however traces the protein component of VLDL and yields different values of VLDL production. These considerations indicate that isotopic tracer techniques have provided an effective mean of studying in-vivo pathways of lipid metabolism in humans. The endogenous labelling of triglycerides by infusion of precursor tracers allows fractional rates of synthesis or catabolism of lipoprotein components to be analysed directly. The other approach involving infusion of intrinsically labelled lipoprotein allows the assessment of the absolute rates of de novo lipoprotein appearance and disappearance as well as inter-conversion of lipoprotein components. Both approaches must be considered therefore complementary. In addition to the production of intrinsically labelled lipoproteins, methods have been also developed to produce intrinsically labelled animal and vegetable proteins for human metabolic studies. Lactating cows were infused with labelled leucine, and milk was collected during the infusion to obtain milk proteins intrinsically labelled with [13C]leucine.7 Casein and whey protein fractions were purified by membrane separation techniques and produced in large amounts with sufficient 13C enrichment to be used in human protein metabolism studies. Intrinsically 15N-labelled vegetable proteins have been recently produced for human metabolic studies.8 Although the administration of labelled precursors in vivo to produce intrinsically labelled macromolecules is undoubtedly a suitable approach to investigate human metabolism according to validated isotopic dilution techniques, the assumption
Editorial
inherent in any tracer dilution method that the pool is homogenous may lead to interpretative limitations. Any heterogeneity between endogenous and exogenous molecules would result in discrimination of tracer and tracee with respect to their metabolic fate.
References 1. Havel RJ, Kane JP, Balasse EO, Segel N, Basso LV. Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest 1970; 49(11):2017–35. 2. Siler SQ, Neese RA, Parks EJ, Hellerstein MK. VLDL-triglyceride production after alcohol ingestion, studied using [2-13C1] glycerol. J Lipid Res 1998;39(12):2319–28. 3. Lemieux S, Patterson BW, Carpentier A, Lewis GF, Steiner G. A stable isotope method using a [(2)H(5)]glycerol bolus to measure very low density lipoprotein triglyceride kinetics in humans. J Lipid Res 1999;40(11):2111–7. 4. Patterson BW, Mittendorfer B, Elias N, Satyanarayana R, Klein S. Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover. J Lipid Res 2002;43(2):223–33. 5. Aarsland A, Chinkes D, Wolfe RR. Contributions of de novo synthesis of fatty acids to total VLDL-triglyceride secretion during prolonged hyperglycemia/hyperinsulinemia in normal man. J Clin Invest 1996;98(9):2008–17. 6. Sidossis LS, Magkos F, Mittendorfer B, Wolfe RR. Stable isotope tracer dilution for quantifying very low density lipoproteintriacylglycerol kinetics in man. Clin Nutr 2004;23(4): this issue, doi:10.1016/j.clnu.2003.11.006. 7. Boirie Y, Dangin M, Gachon P, Vasson MP, Maubois JL, Beaufrere B. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc Natl Acad Sci USA 1997;94:14930–5. 8. Mariotti F, Pueyo ME, Tome D, Mahe S. The bioavailability and postprandial utilisation of sweet lupin (Lupinus albus)-flour protein is similar to that of purified soyabean protein in human subjects: a study using intrinsically 15N-labelled proteins. Br J Nutr 2002;87(4):315–23.
Gianni Bioloa Department of Clinical, Technological and Morphological Sciences, Division of Internal Medicine, University of Trieste, Trieste, Italy E-mail address: [email protected] a
Marcello De Ciccob Department of Anesthesia, Resuscitation, Intensive Care Unit, Centro di Riferimento Oncologico (CRO), Istituto Nazionale Tumori, Aviano, Italy b
www.elsevier.com/locate/clnu
EDITORIAL
Stable isotope tracing and triglyceride kinetics Triglycerides synthesized in the liver are secreted into plasma as a part of very-low density lipoproteins (VLDL). These particles permit the delivery of lipid fuel from the liver to other tissues for energy metabolism. Understanding VLDL-triglyceride kinetics in vivo, in humans, has important physiological and clinical implications. This kind of information will be essential to an understanding of the regulation of plasma lipid transport in the fasting and postprandial states and of the subtle changes that occur in those at risk for coronary heart disease. The paradigm of dyslipidemia in the insulin resistance syndrome associated with visceral obesity as well as type 2 diabetes typically consists of increased plasma levels and flux of VLDL-triglycerides from the liver. A variety of approaches have been used to determine in vivo, in humans, endogenous triglyceride synthesis, each with certain limitations. The direct splanchnic release of triglyceride in VLDL has been determined in humans by measuring the arteriovenous difference in triglyceride concentrations across the splanchnic tissues in combination with splanchnic blood flow.1 This invasive approach provides the most unambiguous information, but precision is low because of the technical problems related to accurate determinations of concentrations and blood flow. The use of isotopic tracers for investigating lipid metabolism in human subjects has increased substantially over the last decade due to marked improvements in analytical technologies and better availability of suitable tracers. The most common isotopic approaches to quantify VLDLtriglyceride kinetics are based on the fact that labelled fatty acids (e.g., palmitate) and glycerol infused into the circulation are readily taken up by the liver to be incorporated into newly synthesized triglycerides. These tracers can be administered as a constant infusion or a bolus injection to determine the monoexponential slopes of the increase2 or of the decline3 in plasma labelled VLDLtriglyceride enrichments, respectively. Compartimental modelling may improve the accuracy of determining VLDL-triglyceride kinetics by account-
ing for tracer recycling.2–4 Recently, the precursor– product approach to determine triglyceride synthesis has been improved by calculating intrahepatic precursor enrichments based on mass isotopomer distribution analysis.2,5 The experiments to determine endogenous triglyceride synthesis based on tracer incorporation are non-invasive and easy to carry out. Nonetheless, the relatively complex mathematical models to derive the kinetic parameters of interest and the uncertainties about the number and location of interacting compartments have made it impossible to obtain reliable and precise quantification of triglyceride kinetics to date. Beside the use of the precursor–product relationship, isotopic tracers can be employed in vivo, in humans, according to the tracer dilution technique. This is a suitable approach to determine appearance in a defined compartment of turning over substrates such as glucose from gluconeogenesis and glycogenolysis, amino acids from protein degradation or glycerol and free fatty acids from triglyceride hydrolysis. This method is conceptually straightforward and does not rely on complicated models and unverifiable assumptions. It requires steady state condition that is easily demonstrable by serial determinations of isotopic enrichments in the accessible compartments. This methods, at least in theory, is also suitable for determination of appearance rates in plasma of large and complex molecules such as proteins and lipoprotein-bound lipids. Nonetheless, a major limitation relies on the fact that pre-labelling of large and complex molecules is technically difficult and involves the risk of intravenous infusion of impure preparations in humans. In this issue of Clinical Nutrition, Sidossis et al. described a novel approach to quantify VLDL-triglyceride production rates in humans according to the traditional isotope dilution technique.6 The method involved the invivo labelling of the glycerol backbone of VLDLtriglyceride with [U-13C3]glycerol, plasmapheresis, isolation of the endogenously labelled VLDL, and autologous reinfusion on a separate occasion. The labelled VLDL-triglycerides were synthesized in vivo
0261-5614/$ - see front matter & 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.clnu.2004.06.002
ARTICLE IN PRESS 440
few days prior to the infusion studies. The authors demonstrated a clear isotopic steady state during the autologous reinfusion of labelled VLDL in both the postabsorptive and fed states. This allowed the use of simple tracer dilution equations for kinetic calculations, thereby avoiding assumptions on the heterogeneity of endogenous VLDL pools. The calculated rate of triglyceride appearance was in good agreement with that obtained by the arteriovenous triglyceride balance across the splanchnic bed.1 Despite the clear advantages of this straightforward approach, the re-injection of biologically labelled plasma VLDL has disadvantages in terms of invasivity and logistics and may be impractical for large scale use. An alternative to the in-vivo prelabelling of VLDL-triglycerides with isotopic tracers of glycerol is the in-vitro iodination of VLDLapoproteins. This procedure however traces the protein component of VLDL and yields different values of VLDL production. These considerations indicate that isotopic tracer techniques have provided an effective mean of studying in-vivo pathways of lipid metabolism in humans. The endogenous labelling of triglycerides by infusion of precursor tracers allows fractional rates of synthesis or catabolism of lipoprotein components to be analysed directly. The other approach involving infusion of intrinsically labelled lipoprotein allows the assessment of the absolute rates of de novo lipoprotein appearance and disappearance as well as inter-conversion of lipoprotein components. Both approaches must be considered therefore complementary. In addition to the production of intrinsically labelled lipoproteins, methods have been also developed to produce intrinsically labelled animal and vegetable proteins for human metabolic studies. Lactating cows were infused with labelled leucine, and milk was collected during the infusion to obtain milk proteins intrinsically labelled with [13C]leucine.7 Casein and whey protein fractions were purified by membrane separation techniques and produced in large amounts with sufficient 13C enrichment to be used in human protein metabolism studies. Intrinsically 15N-labelled vegetable proteins have been recently produced for human metabolic studies.8 Although the administration of labelled precursors in vivo to produce intrinsically labelled macromolecules is undoubtedly a suitable approach to investigate human metabolism according to validated isotopic dilution techniques, the assumption
Editorial
inherent in any tracer dilution method that the pool is homogenous may lead to interpretative limitations. Any heterogeneity between endogenous and exogenous molecules would result in discrimination of tracer and tracee with respect to their metabolic fate.
References 1. Havel RJ, Kane JP, Balasse EO, Segel N, Basso LV. Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest 1970; 49(11):2017–35. 2. Siler SQ, Neese RA, Parks EJ, Hellerstein MK. VLDL-triglyceride production after alcohol ingestion, studied using [2-13C1] glycerol. J Lipid Res 1998;39(12):2319–28. 3. Lemieux S, Patterson BW, Carpentier A, Lewis GF, Steiner G. A stable isotope method using a [(2)H(5)]glycerol bolus to measure very low density lipoprotein triglyceride kinetics in humans. J Lipid Res 1999;40(11):2111–7. 4. Patterson BW, Mittendorfer B, Elias N, Satyanarayana R, Klein S. Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover. J Lipid Res 2002;43(2):223–33. 5. Aarsland A, Chinkes D, Wolfe RR. Contributions of de novo synthesis of fatty acids to total VLDL-triglyceride secretion during prolonged hyperglycemia/hyperinsulinemia in normal man. J Clin Invest 1996;98(9):2008–17. 6. Sidossis LS, Magkos F, Mittendorfer B, Wolfe RR. Stable isotope tracer dilution for quantifying very low density lipoproteintriacylglycerol kinetics in man. Clin Nutr 2004;23(4): this issue, doi:10.1016/j.clnu.2003.11.006. 7. Boirie Y, Dangin M, Gachon P, Vasson MP, Maubois JL, Beaufrere B. Slow and fast dietary proteins differently modulate postprandial protein accretion. Proc Natl Acad Sci USA 1997;94:14930–5. 8. Mariotti F, Pueyo ME, Tome D, Mahe S. The bioavailability and postprandial utilisation of sweet lupin (Lupinus albus)-flour protein is similar to that of purified soyabean protein in human subjects: a study using intrinsically 15N-labelled proteins. Br J Nutr 2002;87(4):315–23.
Gianni Bioloa Department of Clinical, Technological and Morphological Sciences, Division of Internal Medicine, University of Trieste, Trieste, Italy E-mail address: [email protected] a
Marcello De Ciccob Department of Anesthesia, Resuscitation, Intensive Care Unit, Centro di Riferimento Oncologico (CRO), Istituto Nazionale Tumori, Aviano, Italy b