Quantification of fatty acid uptake with [11C]Palmitate PET
Palmitate is a physiological substrate of the heart and other organs, and labelling it with 11C does not change its biochemical properties. Palmitate, like the other non-esterified fatty acids (NEFA), are activated in cytosol to acyl-coenzyme A (acyl-CoA) units by acyl-CoA synthetase, which are then transported into the mitochondria for oxidation, or esterified to neutral lipids and phospholipids. 11C-labelled esterified lipids remain in the tissue during the PET study, except in the liver and intestine, which release the triglycerides (TG) into the circulation.
Quantification of β-oxidation or lipid synthesis in tissue requires the measurement of proper input function, and consideration of 11C carrying metabolites of 11C-palmitate in arterial plasma and in the tissue.
CO2 (and [11C]HCO3-) are produced by peripheral and local metabolism of [11C]palmitate-PET, and it readily enters all tissues and approaches a transient equilibrium based on the difference between tissue and plasma pH (Brooks et al., 1984). Concentration of CO2 in plasma or blood must be measured or estimated and subtracted from the input curve. Also its contribution to tissue radioactivity must be accounted for.
Liver and intestine synthesize triglycerides, containing the labelled 11C-palmitate, and release these to circulation. The appearance of labeled TG represents an additional source of circulating [11C]palmitate since endothelial lipoprotein lipase is present in most peripheral tissues (Guiducci et al., 2006). Therefore the labeled TG concentration in plasma should be included in the input function with the non-esterified 11C-palmitate.
Also 11C bearing urea, lactate and glucose can be found in tissues and blood. All these things considered, full quantification of 11C-palmitate uptake is very demanding, and therefore very simple semiquantitative methods are usually applied.
Heart normally extracts 40-50% of labelled palmitate in a single transit time. The capillary endothelium is a major barrier to the extraction of FFAs.
Myocardial [11C]palmitate PET studies are commonly analyzed by measuring the clearance of [11C] from myocardial regions assuming bi-exponential washout. The faster component correlates with β-oxidation including formation of [11C]-2-acetyl-CoA and production of [11C]CO2 by the tricarboxylic acid (TCA) cycle in the mitochondrial matrix. The slower washout component correlates with incorporation of [11C]palmitate into triglyserides and phospholipids (Schelbert et al., 1986; Grover-McKay et al., 1986).
Bergmann et al. (1996) have developed and validated in dogs a four-compartment model for analysis of myocardial [11C]palmitate PET studies for estimation of myocardial fatty acid utilization (MFAU) and myocardial fatty acid oxidation (FMAO). This method requires that blood TAC is measured, [15O]CO2 appearing in blood is measured and subtracted, and that myocardial blood flow is determined from a separate [15O]H2O scan. This method has been used e.g. by Herrero et al. (2005). Later, de Jong et al. (2009) compared several model settings and concluded that a three-tissue compartment model with three fitted parameters is optimal for analyzing 11C-palmiate data. This model setting was used by Bucci et al. (2011) to estimate free fatty acid esterification, oxidation and uptake rates.
The four-compartment model with five fitted parameters has also been applied to rat studies (Shoghi et al., 2009); method requires that myocardial blood flow is known, and Shoghi et al estimated MBF from a [11C]acetate study; input function was estimated with hybrid-image and blood sampling algorithm (Shoghi et al., 2007).
Angsten et al (2005) analyzed their animal (pig) PET data with a simplified approach: Gjedde-Patlak plot was first used to determine [11C]palmitate net influx rate (KT, or Ki) using arterial curve measured from LV region in the image, and myocardial curve from about half a minute to two minutes after injection. Further, a monoexponential function was fitted to blood curve from about one minute to two minutes after injection and was used to extrapolate the blood curve of parent tracer at later time points. Using this extrapolated blood curve and KT, the total amount of extracted tracer at late times was calculated and compared to the measured tissue concentration at the same time (representing the tracer retained in slow turnover compartment) to calculate the fractional oxidative utilization of palmitate.
Suggested analysis method
Calculate the [11C]palmitate uptake index by dividing the myocardial concentration at 7.5 min (peak value) with the integral of the metabolite corrected plasma curve (Knuuti et al., 2001). Index was then further multiplied with the concentration of non-esterified fatty acids in plasma.
To estimate [11C]palmitic acid β-oxidation rate, a two-exponential function is fitted to the steepest descent of the myocardial curve (11-27.5 min) and the clearance rate (1/min) or half-time (t1/2, min) of the faster component is reported (Knuuti et al., 2001).
Tools for these calculations are available in Carimas.
If compartmental model analysis is preferred, please consult Marco Bucci.
About 50-60% of [11C]palmitate is oxidized to produce [11C]CO2 and other hydrophilic metabolites. The rest enters rapidly the stable brain lipid pool (Miller et al., 1987; Chang et al., 1994). Brain preferentially incorporates fatty acids into phosphoglyserides instead of neutral lipids.
A three-compartment model for palmitate incorporation into (rat) brain has been presented by Tabata et al. (1988). Simplified method for estimating the rate of incorporation of fatty acids into brain phopholipids and other stable compartments (not β-oxidation rate) was further developed in the study of Robinson et al. (1992). Model was also used to analyze monkey brain PET studies (45 min), now including also cerebral blood volume and the brain [11C]CO2 in the model (Arai et al., 1995): mean net uptake rates were about 0.0027 ml(min g)-1 in cortical regions and 0.0017 ml(min g)-1 in white matter. However, monkeys were anesthetized, which in rats reduced brain uptake rate by 40%. Vb was 3.5 - 4.8% in cortical regions and 2.2% in white matter (Arai et al., 1995). The same model was applied to monkey [11C]arachidonic acid studies (Chang et al., 1997). Time delay correction, a different correction method for the brain [11C]CO2 (and [11C]HCO3-, and for partial volume effects were introduced by Giovacchini et al. (2002 and 2004) for [11C]arachidonic acid human PET studies; these methods would be applicable to [11C]palmitate studies.
Esterified re-circulating palmitate seems not to make any measurable contribution to (rat) brain uptake (Purdon et al., 1997).
Suggested analysis method
Time-delay between plasma and tissue TACs must be corrected before analysis using fitdelay. If blood TAC has been measured, it can be corrected for time-delay at the same time.
Preparing the blood curve
Because the fatty acid uptake into the brain is very low, the impact of vascular radioactivity is very high and must be considered in analysis. If blood curve has not been measured, it can be calculated from the total plasma curve (not corrected for metabolites) using p2blood.
Correcting tissue data for [11C]CO2
Before this correction, weights for fitting should be added to the regional tissue data files. This is necessary because the early time frames are usually much shorter (and numerous) than the later frames.
The fractions of [11C]CO2 of the total blood radioactivity has been determined using evaporation method from eight subjects in our laboratory, and the population average of fraction curves and individual total plasma TACs can be used to calculate a good estimate of plasma [11C]CO2 concentration during the PET study using metabcor. This curve is then used as input in calculation of the tissue concentration of [11C]CO2, using one-tissue compartment model and assuming that K1=0.3 mL/(min*mL) and K1/k2=0.43, as suggested by Giovacchini et al. (2002) based on the study of Brooks et al. (1984). This tissue [11C]CO2 curve is then subtracted from the measured regional tissue curves.
Correction for radioactive metabolites in plasma
In the previous phase, as the plasma [11C]CO2 concentration was calculated using metabcor, also the plasma curve corrected for [11C]CO2 was calculated as well.
However, the evaporation method measured only the fraction of [11C]CO2, not the fraction of other labelled metabolites that appear in plasma at later times. In the relatively long brain [11C]palmitate studies also these metabolites (except labeled TG; Guiducci et al., 2006) have to be subtracted from the plasma curve.
One method to correct for these is to fit a single exponential function to the descending part of plasma curve, corrected for [11C]CO2, before the appearance of other labelled metabolites (usually about 15 minutes) and extrapolate the plasma curve after this time using the fitted exponential function; extrapol can be used for this purpose with option -e=15.
Estimation of K
One-tissue compartment model with two parameters, blood volume fraction, VB and a single rate constant, K, or K1, are estimated from the metabolite correct plasma curves, blood curve, and [11C]CO2 corrected and weighted tissue curves using fitk2, version 1.0.1 or later. Specify the following parameter initial values and constraints in a file with option -i=filename :
K1_initial := 0.002
K1_lower := 0
K1_upper := 0.01
K1k2_initial := 0
K1k2_lower := 0
K1k2_upper := 0
Vb_initial := 0.045
Vb_lower := 0
Vb_upper := 0.75
If dynamic PET scan is not possible, then FUR method can be applied (Karmi et al., 2010).
Carimas can be (also) used for doing FUR and K calculations.
Insulin has a strong effect on liver metabolism of [11C]palmitate (Guiducci et al., 2006), suggesting that PET studies with [11C]palmitate and palmitate analogs will provide very interesting insight in regulation of lipid metabolism in liver. Compartmental model has been used to analyze hepatic [11C]palmitate data enabling measurement of hepatic fatty acid uptake, oxidation, storage and triglyceride release rates in pigs and human beings (Iozzo et al., 2010).
More simple analysis methods may also be useful: The results of Yamamura et al. (1998) suggest that the first component of two-exponential function fit could provide useful information on β-oxidation. DeGrado et al. (2000) calculated volume of distribution (VT) from liver PET data, not a [11C]palmitate study, but another tracer with clearly reversible kinetics, and showed that VT (nonlinearly) correlated with [3H]palmitate oxidation rate. Therefore, multiple-time graphical analysis for reversible uptake (Logan plot) with metabolite corrected plasma input might be a usable analysis method for quantitation of [11C]palmitate PET data in liver.
Compartmental model for calculation of fatty acid uptake, esterification and oxidation rates in the skeletal muscle was presented by Bucci et al (2011).
Angsten G, Valind S, Takalo R, Neu H, Meurling S, Långström B. Inhibition of carnitine-acyl transferase I by oxfenicine studied in vivo with [11C]-labeled fatty acids. Nucl. Med. Biol. 2005; 32: 495-503.
Arai T, Wakabayashi S, Channing MA, Dunn BB, Der MG, Bell JM, Herscovitch P, Eckelman WC, Rapoport SI, Chang MC. Incorporation of (1-carbon-11)palmitate in monkey brain using PET. J. Nucl. Med. 1995; 36: 2261-2267.
Bergmann SR, Weinheimer CJ, Markham J, Herrero P. Quantitation of myocardial fatty acid metabolism using PET. J Nucl Med. 1996; 37: 1723-1730.
Brooks DJ, Lammertsma AA, Beaney RP, Leenders KL, Buckingham PD, Marshall J, Jones T. Measurement of regional cerebral pH in human subjects using continuous inhalation of 11CO2 and positron emission tomography. J Cereb Blood Flow Metabol. 1984; 4: 458-465.
Bucci M, Borra R, Någren K, Maggio R, Tuunanen H, Oikonen V, Del Ry S, Viljanen T, Rigazio S, Giannesi D, Parkkola R, Knuuti J, Iozzo P. Human obesity is characterized by defective fat storage and enhanced muscle fatty acid oxidation and trimetazidine gradually counteracts these abnormalities. Am J Physiol Endocrinol Metab. 2011; 301(1): E105-E112.
Bucci M, Borra R, Någren K, Pärkkä J, Del Ry S, Maggio R, Tuunanen H, Viljanen T, Cabiati M, Rigazio S, Taittonen M, Pagotto U, Parkkola R, Opie LH, Nuutila P, Knuuti J, Iozzo P. Trimetazidine reduces endogenous free fatty acid oxidation and improves myocardial efficiency in obese humans. Cardiovasc Therapeutics 2011 (in press).
Chang MCJ, Arai T, Freed LM, Wakabayashi S, Channing MA, Dunn BB, Der MG, Bell JM, Sasaki T, Herscovitch P, Eckelman WC, Rapoport SI. Brain incorporation of [1-11C]arachidonate in normocapnic and hypercapnic monkeys, measured with positron emission tomography. Brain Res. 1997; 755: 74-83.
Chang MC, Wakabayashi S, Bell JM, Rapoport SI. The effect of methyl palmoxirate on incorporation of [U-14C]palmitate into rat brain. Neurochem Res. 1994; 19(9): 1217-1223.
DeGrado TR, Wang S, Rockey DC. Preliminary evaluation of 15-[18F]fluoro-3-oxa-pentadecanoate as a PET tracer of hepatic fatty acid oxidation. J Nucl Med. 2000; 41(10): 1727-1736.
de Jong HWAM, Rijzewijk LJ, Lubberink M, van der Meer RW, Lamb HJ, Smit JWA, Diamant M, Lammertsma AA. Kinetic models for analysing myocardial [11C]palmiate data. Eur J Nucl Med Mol Imaging 2009; 36: 966-978.
Giovacchini G, Chang MCJ, Channing MA, Toczek M, Mason A, Bokde ALW, Connolly C, Vuong B-K, Ma Y, Der MG, Doudet DJ, Herscovitch P, Eckelman WC, Rapoport SI, Carson RE. Brain incorporation of [11C]arachidonic acid in young healthy humans measured with positron emission tomography. J Cereb Blood Flow Metab. 2002; 22: 1453-1462.
Giovacchini G, Lerner A, Toczek MT, Fraser C, Ma K, DeMar JC, HerscovitchP, Eckelman WC, Rapoport SI, Carson RE. Brain incorporation of 11C-arachidonic acid, blood volume, and blood flow in healthy aging: a study with partial-volume correction. J Nucl Med. 2004; 45: 1471-1479.
Grover-McKay M, Schelbert HR, Schwaiger M, Sochor H, Guzy PM, Krivokapich J, Child JS, Phelps ME. Identification of impaired metabolic reserve by atrial pacing in patients with significant coronary artery stenosis. Circulation 1986; 74(2): 281-292.
Guiducci L, Järvisalo M, Kiss J, Någren K, Viljanen A, Naum AG, Gastaldelli A, Savunen T, Knuuti J, Salvadori PA, Ferrannini E, Nuutila P, Iozzo P. [11C]palmitate kinetics across the splanchnic bed in arterial, portal and hepatic venous plasma during fasting and euglycemic hyperinsulinemia. Nucl Med Biol. 2006; 33(4): 521-528.
Herrero P, Soto PF, Dence CS, Kisrieva-Ware Z, Delano DA, Peterson LR, Gropler RJ. Impact of hormone replacement on myocardial fatty acid metabolism: potential role of estrogen. J Nucl Cardiol. 2005; 12(5): 574-581.
Iozzo P, Bucci M, Roivainen A, Någren K, Järvisalo MJ, Kiss J, Guiducci L, Fielding B, Naum AG, Borra R, Virtanen K, Savunen T, Salvadori PA, Ferrannini E, Knuuti J, Nuutila P. Gastroenterology. 2010; 139(3): 846-56, 856.e1-6. Epub 2010 May 25.
Karmi A, Iozzo P, Viljanen A, Hirvonen J, Fielding BA, Virtanen K, Oikonen V, Kemppainen J, Viljanen T, Guiducci L, Haaparanta-Solin M, Någren K, Solin O, Nuutila P. Increased brain fatty acid uptake in metabolic syndrome. Diabetes 2010; 59: 2171-2177.
Knuuti J, Takala TO, Någren K, Sipilä H, Turpeinen AK, Uusitupa MIJ, Nuutila P. Myocardial fatty acid oxidation in patients with impaired glucose tolerance. Diabetologia 2001; 44: 184-187.
Miller JC, Gnaedinger JM, Rapoport SI. Utilization of plasma fatty acid in rat brain: distribution of [14C]palmitate between oxidative and synthetic pathways. J Neurochem. 1987; 49(5): 1507-1514.
Purdon D, Arai T, Rapoport S. No evidence for direct incorporation of esterified palmitic acid from plasma into brain lipids of awake adult rat. J Lipid Res. 1997; 38(3): 526-530.
Robinson PJ, Noronha J, DeGeorge JJ, Freed LM, Nariai T, Rapoport SI. A quantitative method for measuring regional in vivo fatty-acid incorporation into and turnover within brain phospholipids: review and critical analysis. Brain Res Brain Res Rev. 1992; 17(3): 187-214.
Schelbert HR. PET contributions to understanding normal and abnormal cardiac perfusion and metabolism. Ann Biomed Eng. 2000; 28(8): 922-929.
Schelbert HR, Henze E, Sochor H, Grossman RG, Huang S-C, Barrio JR, Schwaiger M, Phelps ME. Effects of substrate availability on myocardial C-11 palmitate kinetics by positron emission tomography in normal subjects and patients with ventricular dysfunction. Am Heart J. 1986; 111: 1055-1064.
Shoghi KI, Finck BN, Schechtman, Sharp T, Herrero P, Gropler RJ, Welch MJ. In vivo metabolic phenotyping of myocardial substrate metabolism in rodents: differential efficacy of metforming and rosiglitazone monotherapy. Circ Cardiovasc Imaging 2009; 2: 373-381.
Shoghi KI, Welch MJ. Hybrid image and blood sampling input function for quantification of small animal dynamic PET data. Nucl Med Biol. 2007; 34: 989-994.
Tabata H, Kimes AS, Robinson PJ, Rapoport SI. Stability of brain incorporation of plasma palmitate in unanesthetized rats of different ages, with appendix on palmitate model. Exp. Neurol. 1988; 102: 221-229.
Tamaki N, Kawamoto M, Takahashi N, Yonekura Y, Magata Y, Torizuka T, Nohara R, Kambara H, Konishi J. Assessment of myocardial fatty acid metabolism with positron emission tomography at rest and during dobutamine infusion in patients with coronary artery disease. Am. Heart J. 1993; 125(3): 702-710.
Yamamura N, Magata Y, Kitano H, Konishi J, Saji H. Evaluation of [1-11C]octanoate as a new radiopharmaceutical for assessing liver function using positron emission tomography. Nucl. Med. Biol. 1998; 25: 467-472.