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Very low density lipoproteins (VLDL) are of interest for two reasons. First, they are the obligate precursor of low density lipoproteins (LDL) (Dietschy et al. 1993). Second, together with chylomicrons, VLDLs represent the major vehicle for transport of triglycerides in the circulation. VLDL triglyceride secretion by the liver is controlled to some extent by the supply of FFA to that tissue (Lewis et al. 1995), with additional acute regulation by insulin (Sparks & Sparks 1994).

Hepatic VLDL production can be determined using several different techniques. One of the first methods utilised by investigators was the hepatic balance technique (Basso & Havel 1970). This approach requires arterial, portal vein and hepatic vein sampling, together with a measurement of hepatic blood flow, and thus can only be used in animal studies. Splanchnic triglyceride balance (sampling arterial and hepatic venous blood, without portal venous sampling) has been measured in humans (Havel et al. 1970). The procedure is reasonably safe in experienced hands. However, it likely produces underestimates of actual hepatic VLDL triglyceride secretion rates because of uptake of VLDL triglyceride from arterial blood by nonhepatic splanchnic tissues, primarily visceral adipose tissue. Tracer techniques are far preferable to splanchnic balance measurements because they avoid this pitfall and in addition are noninvasive.

One of the first attempts to apply such a tracer technique was a study by Reaven et al. (1965). The investigators determined plasma triglyceride kinetics in normotriglyceridemic and hypertriglyceridemic subjects after an overnight fast using a bolus injection of radiolabeled glycerol, and measured tracer disappearance for eight hours after the injection. VLDL was isolated by ultracentrifugation and triglycerides were subsequently isolated using thin layer chromatography. Using a two-compartmental model for data analysis, the hypertriglyc-eridemic subjects had markedly higher triglyceride production rates and markedly lower fractional catabolic rates compared with the normotriglyceridemic subjects. Subsequently, numerous investigators published studies using a similar technique, in some instances using a fatty acid tracer instead of a glycerol tracer to label VLDL (Quarfordt et al. 1970).

Perhaps the most detailed investigation of tracer methodology for VLDL kinetics was conducted by Patterson et al. (2002), who administered a bolus of [2H5] glycerol (16 subjects) or [13C] palmitate (7 subjects) to normotriglyceridemic subjects and applied both monoexponential and compartmental analysis to the data. An additional five subjects received a constant infusion of [13C] palmitate to determine turnover from the rise-to-plateau of VLDL triglyceride enrichment, as described by Parks et al. (1999). Laboratory analyses were performed using gas chromatography/mass spectrometry (GC/MS). Fractional turnover was slower with the fatty acid tracer than with glycerol. When kinetics of an endogenous substance are determined by administering a labeled precursor, calculations are based on the disappearance of the labeled product. If there is ongoing production of that product during the time that disappearance is being measured, the slope of disappearance will be less steep and an error will be introduced. In the case of VLDL, the precursor (glycerol or a fatty acid) enters the intrahepatic VLDL triglyceride pool and resides there for a finite period of time before secretion into the circulation. Previous studies have shown that the systemic clearance of glycerol from plasma is 3 to 4-fold higher than that of FFA (Coppack et al. 1999). If the intrahepatic clearance of glycerol is similarly greater than that of fatty acids, then intrahepatic labeled glycerol disappears more rapidly than intrahepatic labeled palmitate, making it unavailable for further VLDL triglyceride synthesis. This may explain why a fatty acid tracer provides lower estimates of VLDL turnover than a glycerol tracer.

Patterson et al. (2002) also found that compartmental modeling produced an estimate of fractional VLDL turnover that was nearly double the value obtained with monoexponential analysis. The discrepancy was even greater when compared with previous reports using [3H] glycerol, whether or not compartmental analysis was used. This could indicate greater tracer recycling with [3H] glycerol compared to [2H5] glycerol, or could simply be related to the fact that different subjects were studied and fractional turnover varies among subjects. Another possibility, however, relates to isotope effects, which are generally more of a problem with hydrogen labeled tracers than with carbon labeled tracers. The potential for isotope effects can be greater for tritium than for deuterium (Northrop 1981). In general, an isotope effect results in a slowing of enzyme-mediated reactions, and thus would produce slower flux rates, lower clearance and lower fractional turnover.

Whether glycerol or a fatty acid tracer is used, high quality laboratory analyses are important. Older methods for VLDL kinetics analysed tracer and tracee in separate procedures (Reaven et al. 1965; Quarfordt et al. 1970). This adds to analytical imprecision and can be a limiting factor. When HPLC (Miles et al. 1987; Judd et al. 1998) or GC/MS (Guo et al. 1997; Gilker et al. 1992) are used, the measurement of tracer and tracee are linked, minimising imprecision. This is particularly important when conducting extended sampling, as is needed for compartmental analysis. An example of VLDL disappearance curves (in this case using [2H5] glycerol as a precursor) is shown in Figure 9.5. As can be seen, [2H5] enrichment in VLDL triglyceride peaked later in individuals with type 2 diabetes than in control subjects (Isley et al. 2006). The explanation for this is not clear, but it could represent slower fractional turnover of either intrahepatic VLDL triglyceride or its precursor fatty acid pool.

A limitation in many studies of lipoprotein kinetics has been the use of a precursor to label a product of interest, as discussed above. There have been attempts to develop techniques

Tracer Techniques Biology

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Figure 9.5 Isotopic enrichment curves in normal subjects (diamonds) and in type 2 diabetic subjects before (triangles) and after (squares) 12 weeks of therapy with high dose (80 mg) simvastatin. TTR = tracer:tracee ratio. Reproduced from Isley WL, Miles JM, Patterson BW, Harris WS (2006) The effect of highdose simvastatin on triglyceride-rich lipoprotein metabolism in patients with type 2 diabetes mellitus. J Lipid Res 47:193-200. Courtesy of The American Society for Biochemistry and Molecular Biology.

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Figure 9.5 Isotopic enrichment curves in normal subjects (diamonds) and in type 2 diabetic subjects before (triangles) and after (squares) 12 weeks of therapy with high dose (80 mg) simvastatin. TTR = tracer:tracee ratio. Reproduced from Isley WL, Miles JM, Patterson BW, Harris WS (2006) The effect of highdose simvastatin on triglyceride-rich lipoprotein metabolism in patients with type 2 diabetes mellitus. J Lipid Res 47:193-200. Courtesy of The American Society for Biochemistry and Molecular Biology.

for labeling VLDL for subsequent intravenous administration. Two potential approaches have been used. A radiolabeled glycerol or fatty acid can be administered, and VLDL can beharvested from the plasma 1-2 hours later for subsequent use. Wolfe et al. (1985) used this technique to obtain labeled VLDL from donor dogs for subsequent infusion into other animals in a study of VLDL kinetics in sepsis. Sidossis et al. (2004) gave oral doses of [U-13C] glycerol to normal volunteers and harvested labeled VLDL by plasmapheresis. The labeled VLDL was administered using a two-hour constant infusion 2-3 days later. The infusion was 'primed' with a bolus dose equal to three times the hourly infusion rate. Fractional turnover rates were 1.92.2 pools/h after an overnight fast on normal and high fat diets, and somewhat lower (1.31.7 pools/h) on a high fat diet and during a glucose infusion. Considering the relatively long half-life of VLDL triglyceride in plasma, the adequacy of the isotopic steady state achieved in this study is not entirely clear, since samples were limited to a 20 minute interval 100-120 minutes after the start of the infusion. However, this report represents a major advance in the development of a novel technique for the measurement of VLDL kinetics in humans.

Early investigators harvested VLDL from study subjects and prepared a radioiodinated VLDL ex vivo, followed by filter sterilisation prior to autologous use (Sigurdsson et al. 1975). Studies such as this suggest that ex vivo labeling of lipoproteins might be feasible, with the same caveats about sterility. However, use of radioiodinated tracers has fallen from favour because of the radiation exposure involved, in addition to concerns that iodination alters the metabolism of the lipoprotein particle. Recently, Gormsen et al. (2006a) reported a technique for ex vivo preparation of a VLDL tracer using a radiolabeled triglyceride

(triolein) for subsequent autologous use. It was necessary to sonicate the material at 37 °C for six hours in order to fully incorporate the radiolabeled triglyceride into VLDL. When tracer thus prepared was infused into the subject, very little spillover of LPL-generated fatty acids was observed. This is consistent with the work of Wolfe et al. (1985), who found <5% fractional spillover of endogenously labeled VLDL in dogs. The use of an ex vivo labeled VLDL tracer avoids the difficulties presented when a labeled precursor is given and the VLDL disappearance curve is altered by ongoing secretion of labeled VLDL. In addition, the low rates of spillover observed indicate minimal potential for tracer recycling.

Gormsen et al. (2006b) have reported additional VLDL kinetic data in 25 normotriglyceridemic subjects using a modification of this technique. Sampling for five hours and using monoexponential analysis, they found VLDL fractional turnover of ~0.25 pools/h. This is strikingly different than the results of Patterson et al. (2002), who reported fractional turnover rates of 0.64 and 1.06 pools/h using monoexponential and compartmental analysis, respectively, with the [2H5] glycerol method. The implications of this are not entirely clear. Individuals with higher fractional turnover would be expected to have lower plasma triglyceride concentrations (Reaven et al. 1965). Comparison of the two studies is difficult, especially considering that Patterson found essentially identical results with monoexponential and compartmental analysis when fractional turnover was <0.4 pools/h. It is possible that tracer recycling, in relative terms, creates a larger error in the measurement when fractional VLDL turnover is high and half life is short, at least when a precursor labeling method is used. Significant enrichment in plasma glycerol one and two hours after the injection of labeled glycerol, as shown by Patterson et al. (2002), would contribute a greater error to apparent monoexponential disappearance of VLDL when VLDL fractional turnover is higher, because of a greater contribution on ongoing synthesis and secretion of labeled VLDL during the first several hours after the injection to VLDL triglyceride enrichment/specific activity. Recycling of 2H or 3H through the body-water pool would be an unlikely factor because of the enormous dilution that would be expected to occur with recycling via this pathway. Ex vivo labeling has theoretical advantages over in vivo labeling, as discussed above. However, additional studies of the ex vivo method, in which it is directly compared with the [2H5] glycerol method, and perhaps (if feasible) extended sampling and compartmental analysis, are warranted. A comparison of the two methods using only monoexponential analysis in individuals with low (say <0.8 mmol/L) plasma total triglyceride concentrations would be particularly informative, since these subjects would be expected to have higher fractional turnover rates. In addition to a need for further validation experiments, any method that involves tracer preparation in the laboratory, including isolation of biosynthetic tracer (Sidossis et al. 2004), obviously requires scrupulous sterile technique and safeguards against contamination.

A limitation of the methods that involve benchtop processing of an autologous VLDL tracer is that the material is reinfused within a relatively short period of time - 2-7 days (Sidossis et al. 2004; Gormsen et al. 2006a, 2006b). If a method that would allow long term storage of a biosynthesised or ex vivo prepared tracer were available, the tracer could be collected under baseline conditions (e.g. before weight loss or before drug therapy), then administered after the intervention. Plasmapheresis and subsequent cryopreservation of tracer has been applied successfully to low density lipoproteins (Rumsey et al. 1992, 1994) and could potentially be used to administer VLDL tracer several months after harvesting.

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