Tracers for the study of triglyceriderich lipoprotein kinetics Chylomicrons

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Chylomicrons are large lipoprotein particles that are formed by the small intestine during fat absorption. The amount of lipid fuel that traverses the circulation in the form of chylomicrons obviously varies with dietary fat consumption. In individuals on high-fat diets, however, it can equal or exceed FFA flux (Miles et al. 2004). The triglyceride transported in chylomicrons is metabolised by LPL, which is widely distributed in tissues but is most abundant in adipose tissue and skeletal muscle (Eckel 1989). Nascent chylomicrons have a short residence time in the circulation, with a half life of approximately five minutes (Park et al. 2001). Chylomicron half life is prolonged at higher chylomicron triglyceride concentrations (Park et al. 2001).

Triglyceride uptake from the circulation can under some circumstances occur independent of enzymes such as LPL and hepatic lipase. Saturation of transport appears to occur at plasma triglyceride concentrations of ~400mg/dL-1 (Brunzell et al. 1973, 1979) or lower (Nikkila & Kekki 1973). It is important to note that chylomicron-sized lipid particles can be removed via nonenzymatic pathways, particularly the reticuloendothelial system (Seidner et al. 1989). When large amounts of a lipid emulsion were administered to rats by bolus injection, there was evidence of non-enzymatic lipid clearance (Lutz et al. 1989). Karpe et al. (1997) concluded that, during mild chylomicronaemia after a high fat meal in normal subjects, the removal of triglycerides by non-lipolytic tissues was negligible. Quantitatively significant reticuloendothelial uptake may occur only at plasma triglyceride concentrations above those at which maximal rates of LPL-mediated triglyceride hydrolysis are observed, i.e. ~300-400 mg/dL-1 or 3.4-4.5 mmol/L-1 (Miles et al. 2001).

Mere measurement of triglyceride concentrations (and triglyceride-rich lipoprotein concentrations) in plasma cannot distinguish defects in production from abnormalities in removal and provide no insight into kinetic events. Many investigators have therefore utilised experimental procedures for this purpose. A variety of techniques, using native chylomicrons, lipid emulsions as a surrogate for chylomicrons, and both labeled and unlabeled materials, have been employed.

The choice of method for tracking the metabolism of chylomicrons depends substantially on the intended focus of the investigation: the triglyceride fatty acids in the nascent particle or the particle itself, together with its remnants. There is substantial evidence to suggest that chylomicron remnants are atherogenic (Redgrave 2004). An older method for studying chylomicron particle metabolism involved labeling of a fat meal with retinol palmitate (Hazzard & Bierman 1976). However, the fact that retinol palmitate appears in very low density lipoprotein (VLDL) particles (Lemieux et al. 1998) and that it is hydrolysed and taken up by adipose tissue LPL (Blaner et al. 1994) indicates that it is not an ideal tracer for this purpose. An alternative approach suitable for tracing the metabolism of chylomicron remnants is the labeling of apolipoprotein B-48 with a stable isotope (Lichtenstein et al. 1992). Finally, labeled cholesterol esters can be added to a synthetic lipid emulsion and administered intravenously (Redgrave & Maranhao 1985). The cholesterol ester disappears much more slowly from plasma than does labeled triglyceride (Redgrave & Maranhao 1985), as would be expected with a tracer that is retained in the particle after its interaction with LPL.

The fate of dietary fatty acids can be traced by adding a radiolabeled triglyceride to a mixed meal and tracking the appearance of the labeled fatty acid in the plasma space and its subsequent uptake in regional fat depots (Roust & Jensen 1993; Romanski et al. 2000; Jensen et al. 2003). Triglycerides or fatty acids labeled with stable isotopes can be administered as part of a meal in order to generate labeled chylomicrons (Evans et al. 2002; Barrows et al. 2005). The technique can be extremely useful in assessing patterns of dietary fat storage. It has the advantage that the secreted chylomicrons contain physiological mixed triglycerides. A limitation is that tracer input (i.e. the rate of absorption of labeled chylomicrons) is unknown.

Nestel et al. (1962) administered chylomicrons harvested surgically from thoracic duct lymph to normal volunteers to study triglyceride-rich lipoprotein metabolism. Whereas this represents a physiological approach, the invasiveness of the method limits its usefulness. Because of such limitations, investigators have since then employed a lipid emulsion as a surrogate for native chylomicrons. Although some investigators have used a bolus injection of unlabeled lipid (Rossner 1974), this also has limitations because it perturbs the same intrinsic parameters (clearance; half life; residence time) that the technique is attempting to measure, increasing plasma triglyceride concentrations to >1000 mg/dL-1 (11.3 mmol/L-1). In fact, triglyceride clearance estimated from intravenous fat tolerance tests is 100-200 % slower (Rossner 1974; Cohen 1989) than the clearance of a tracer dose of radioactive lipid (Nakandakare et al. 1994). Moreover, the intravenous fat tolerance test may result in significant, if brief, reticuloendothelial uptake of lipid because of the temporary high triglyceride concentrations achieved, as described above. A bolus injection of a radiolabeled lipid emulsion of relatively high specific activity (Nakandakare et al. 1994), on the other hand, allows the investigator to give what is in essence a massless amount of lipid as a tracer.

One of the key questions with this experimental approach is whether the artificial lipid emulsion is metabolised in a fashion that approximates that of chylomicrons. Infused lipid particles rapidly acquire apolipoprotein C2 (Iriyama et al. 1988). However, this does not ensure that the emulsion particles are metabolised similarly to chylomicrons. It is clear that the absence of cholesterol and cholesterol esters in artificial lipid emulsions results in the in vivo production of remnants that are cleared much more slowly than chylomicron remnants. To make it possible to trace chylomicron remnant metabolism with a radiolabeled artificial lipid emulsion, Redgrave developed (Redgrave & Maranhao 1985) and validated (Redgrave et al. 1993) a technique in which cholesterol and cholesterol esters are incorporated in chylomicron-like particles, and radiolabeled phospholipid-rich vesicles are removed by ultracentrifugation. This method allows the investigator to trace chylomicron triglyceride and remnant particle metabolism by adding radiolabeled triglyceride and cholesterol ester, respectively, during emulsion preparation, and produces clearance data that are very similar to results with chylomicrons (Redgrave & Maranhao 1985; Redgrave et al. 1993). From available data, it appears that a chylomicron-like particle requires cholesterol and cholesterol esters in order to undergo normal remnant metabolism, but these components have little effect on particle interaction with LPL, based on the observations of Redgrave & Maranhao (1985).

For in vivo investigations, a tracer-containing lipid emulsion can be prepared from oil, water, an emulsifying agent and other ingredients using sonication and density grandient ultracentrifugation. This approach has been used for studies in rodents (Redgrave & Maranhao 1985; Redgrave et al. 1993) and in humans (Redgrave et al. 1993; Nakandakare et al. 1994). Alternatively, a radiolabeled triglyceride can be incorporated into a commercial lipid emulsion (Park et al. 2000). Although triolein is not an entirely physiological triglyceride, most investigators (Redgrave & Maranhao 1985; Redgrave et al. 1993; Nakandakare et al. 1994; Hultin et al. 1995; Martins et al. 1996; Park et al. 2000) have found that emulsions labeled with this material provide good estimates of chylomicron triglyceride metabolism, with no evidence of exchange of labeled triolein with other lipoproteins when administered to humans (Nakandakare et al. 1994).

When labeled triolein is added to a commercial lipid emulsion, it is distributed roughly equally between large triglyceride-rich, chylomicron-sized (mean diameter ~340 nm) particles and much smaller phospholipid-rich vesicles (Park et al. 2000). A size exclusion HPLC purification step is therefore required to isolate the large particles in pure form; a radiochro-matogram showing the separation of the two species of particles is shown in Figure 9.3. The material is immediately collected in a vial containing unlabeled lipid emulsion, which maintains its stability by restoring the phospholipid excess present in the commercial preparation, and is then autoclaved. The particle size spectrum of this preparation is almost identical to that of native chylomicrons (Park et al. 2000), as shown in Figure 9.4.

The labeled lipid emulsion prepared in this fashion appears to be stable for at least a month (unpublished results). Infused at a rate of 0.6-1.2 ^Ci/min, it has a sufficiently high specific activity (~400,000 dpm/^mol fatty acid) that the infusion does not result in an increase in plasma triglyceride concentrations. Limitations on the amount of material that can be incorporated into the emulsion preclude using this technique to prepare an emulsion labeled with a stable isotopic tracer. Isolation of chylomicrons from plasma, separate from particles containing apolipoprotein B-100, is a challenge. A reasonably pure chylomicron isolate can be obtained with triple ultracentrifugation, although it still contains a small (~4 % of total triglyceride) contribution from VLDL (Park et al. 2000). This indicates that roughly half of the particles in the specimen are VLDL, and half are chylomicrons. Immunoaffinity

minutes

Figure 9.3 A size exclusion HPLC radiochromatogram of unpurified lipid emulsion (solid line) and a rechromatographed, purified 30-40 min fraction containing chylomicron sized particles (dashed line). Reproduced courtesy of Park Y, Grellner WJ, Harris WS, Miles JM, (2000) A new method for the study of chylomicron kinetics in vivo. Am J Physiol 279, E1258-E1263.

Figure 9.3 A size exclusion HPLC radiochromatogram of unpurified lipid emulsion (solid line) and a rechromatographed, purified 30-40 min fraction containing chylomicron sized particles (dashed line). Reproduced courtesy of Park Y, Grellner WJ, Harris WS, Miles JM, (2000) A new method for the study of chylomicron kinetics in vivo. Am J Physiol 279, E1258-E1263.

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