Kinetics of an Intravenously Injected Fat Emulsion Reflect Postheparin Plasma Lipoprotein Lipase Mass and Activity

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1 J. Clin. Biochem. Nut:, 31, 37-43, 2002 Kinetics of an Intravenously Injected Fat Emulsion Reflect Postheparin Plasma Lipoprotein Lipase Mass and Activity Nagahiko SAKUMA,* Reiko IKEUCHI, Takeshi HIBINO, Nobuyuki OHTE, Nozomu TAMAI, Takeshi KAMIYA, and Genjirou KIMURA The Third Department of Internal Medicine, Nagoya City University Medical School, Nagoya , Japan (Received January 15, 2002) Summary A study was conducted to determine whether the catabolism of an intravenously injected fat emulsion could be correlated with lipoprotein lipase (LPL) mass and activity in postheparin plasma. We observed the relationship between the fractional removal rate (K2) of an intravenously injected fat emulsion (Intralipid ) and LPL mass and activity in postheparin plasma of 20 normolipidemic and 21 hypertriglyceridemic subjects. The K2 level was determined by the intravenous fat emulsion tolerance test. LPL mass was measured by a sandwich enzyme immunoassay system. K2 was significantly correlated with the LPL mass in total group of subjects (r=0.51, p<0.001), as well as in normolipidemic (r=0.72, p<0.001) and hypertriglyceridemic (r=0.43, p<0.05) subgroups. K2 was also significantly correlated with LPL activity in the total group (r=0.40, p<0.05), as well as in the normolipidemic subjects (r=0.55, p<0.05). Kinetics following an intravenous injection of a fat emulsion reflect LPL mass and activity in postheparin plasma. Key Words: fat emulsion kinetics, lipoprotein lipase in postheparin plasma, Intralipid(R), fat emulsion tolerance test, triglyceride Hypertriglyceridemia, a risk factor for coronary heart disease [1], may be related to a decelerated catabolism or accelerated synthesis of serum triglycerides (TG), or both. A simple intravenous fat emulsion tolerance test using Intralipid (Vitrum AB, Stockholm) can be used to assess abnormal TG removal in patients with hypertriglyceridemia, since the catabolic capacity of serum TG can be indirectly estimated from the fractional removal rate of Intralipid (Kz, %1 min) using this test [2-61. Intralipid, an artificial fat emulsion used clinically to supply energy and essential fatty acids, is similar in characteristics to chylomicrons in terms of TG content, particle *To whom correspondence should be addressed. The Third Department of Internal Medicine, Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya , Japan (Tel.: ; Fax.: ). 37

2 38 N. SAKUMA et al. size, and the metabolic pathways. After injection into the bloodstream, the artificial fat emulsion accepts apolipoprotein E, C-II and C-III from high-density lipoprotein (HDL), and is then metabolized via first-order kinetics by lipoprotein lipase (LPL) [7, 8]. LPL hydrolyzes triglycerides in circulating chylomicrons and very low-density lipoprotein (VLDL) on the surface of endothelial cells [9, 10]. This enzyme is thought to be synthesized mainly in adipose tissues and muscles, and to be then transported to the luminal surface of endothelial cells [11]. LPL binds to the cell surface by means of proteoglycans via electrostatic interactions [12]. The injection of heparin causes release of LPL from the endothelial surface [13]. However, it is reported that preheparin plasma contains substantial amounts of inactive LPL protein and associated with cholesterol-rich lipoproteins, and that heparin releases mainly active LPL into circulation. Also, it is reported that there was a strong correlation between the increments of LPL mass and activity in postheparin plasma [14] and that there was a strong correlation between LPL mass and activity after heparin, supporting the view that heparin releases the active form of enzyme [15]. Meanwhile, it is reported that LPL mass in preheparin plasma correlated negatively with TG and positively with HDL-cholesterol. These results are consistent with the idea that preheparin LPL mass could reflect some amount of working LPL in the whole body, because LPL itself hydrolyzes TG, resulting in a decrease of serum TG level and an increase of HDL-cholesterol [16]. The implication of these findings is that there are two parameters to measure with postheparin plasma, the amount of LPL mass and LPL activity. To determine whether the catabolism of an intravenously injected fat emulsion might reflect LPL mass and activity in postheparin plasma, we investigated correlations between KZ and LPL mass and activity in postheparin plasma of normolipidemic and hypertriglyceridemic subjects. MATERIALS AND METHODS We evaluated 41 Japanese subjects (22 men and 19 women; mean age±sd, 39±13 years) from the outpatient clinic of the Third Department of Internal Medicine, Nagoya City University Hospital, Nagoya, Japan after obtaining their informed consent. Of these 41 subjects, 20 were normolipidemic and 21 were hypertriglyceridemic. Normolipidemia was defined as a serum TG concentration below 150 mg/dl and a serum total cholesterol (TC) concentration below 220 mg/dl. The fat emulsion tolerance test was performed after blood samples have been collected on fasting for 14 h in the morning for measurement of serum lipids. Two hours later, blood samples were obtained before and 10 min after rapid intravenous administration of heparin sodium (30IUlkg body weight) for measurement of plasma LPL mass and activity. Serum TG and TC concentrations were measured by enzymatic methods. The postheparin plasma LPL mass was measured with an LPL kit (MARKIT F LPL; Dainippon Pharmaceutical Co., Ltd., Osaka) using a sandwich-enzyme immunoassay system according to the manufacturer's instructions [17]. The postheparin plasma LPL activity was J. Clin. Biochem. Nuts

3 FRACTIONAL REMOVAL RATE OF FAT EMULSION AND LIPOPROTEIN LIPASE 39 measured according to the method of Nozaki et al. [18]. The fat emulsion tolerance test [2] was performed as follows. Intralipid containing a 10% fat emulsion (0.25 ml/kg body weight) was administered intravenously over 90 s. The midpoint of the intravenous administration (at 45 s) was defined as the zero time point, and blood samples were collected at 3, 5, 7, 9, 11, 14, 17, and 20 min. Thereafter, each blood sample was centrifuged at 4 C and 1,800Xg for 30 min to obtain the serum. Serum samples obtained at every time point were divided into 2 portions, and each portion was diluted 100-fold with physiological saline. The light-scattering index (LSI) was determined for each sample with a nephelometer (model DN-2110, Kyoto Daiichi Kagaku, Kyoto). The wavelength used is 610 nm or greater. The LSI is expressed in terms of the value obtained with a special optical glass standard equipped. Using the logarithmic value of the mean LSI of samples at every time point minus the mean LSI of samples obtained before the fat emulsion loading, we calculated the regression coefficient (-b) of a steadily declining LSI curve by the method of least squares, and this value was used to indicate the fractional removal rate (K2) of the fat emulsion (10% Intralipid ). As an index of the reliability of b, Student's t value was obtained from b/sb (Sb representing the standard error of b). The reliability of K2 can be determined from the t-value. If t-value is greater than 5, the confidence interval of K2 is considered to be small. The t-values of K2 of the subjects in this study were greater than 5. RESULTS Table 1 shows serum lipid concentrations, K2 values, and LPL mass and activity levels in postheparin plasma of normolipidemic and hypertriglyceridemic subjects. Each serum lipid concentration of the hypertriglyceridemic subjects was significantly higher than that of the normolipidemic subjects (p<0.01). K2 was significantly correlated with the LPL mass in the total group of subjects (r=0.51, p<0.001) as well as in the normolipidemic (r=0.72, p<0.001) and hypertriglyceridemic subgroups (r=0.43, p<0.05) (Fig. 1). Similarly, K2 was significantly correlated with the LPL activity in the total group of subjects (r=0.40, p<0.05) and in the subgroup of normolipidemic subjects (r=0.55, Table 1. Serum lipid levels, K2 values, and postheparin plasma LPL mass and activity levels in all subjects, normolipidemic subjects, and hypertriglyceridemic subjects. Mean ±SD (range); normolipidemic subjects vs. hypertriglyceridemic subjects: *p<0.05, **p<0.01. TO, triglyceride; T-ch, total-cholesterol; HDL-ch, HDL-cholesterol; LPL mass, postheparin plasma LPL mass (ng/ml); LPL activity, postheparin plasma LPL activity (µmol/ml/min) Vol. 31, 2002

4 40 N. SAKUMA et al. Fig. 1. Correlation between the fractional removal rate of fat emulsion (K2) and protein level of postheparin lipoprotein lipase. 0, Normolipidemic subjects;, hypertriglyceridemic subjects. p<0.05). Kz was not significantly correlated with the plasma LPL activity in hypertriglyceridemic subjects. DISCUSSION Weinberg and Scanu reported a net transfer in vitro of cholesterol esters from HDL to Intralipid, with a reciprocal net transfer of TG to HDL [19]. After Intralipid, an artificial fat emulsion, is injected into the bloodstream, it is metabolized by LPL in the same manner in which chylomicrons are metabolized [8]. When Intralipid enters the circulation, it does not contain the small molecular weight apolipoprotein C, such as apo C-II which is probably the sole activator of LPL [20], which in turn hydrolyzes circulating TG. Havel et al. demonstrated that Intralipid acquires C peptides from HDL, and thereby becomes a suitable substrate for LPL [21]. Ultra-histochemical studies have shown that, as with chylomicrons, Intralipid TG are hydrolyzed at the surface of endothelial cells in capillaries [22, 23]. The fat emulsion Intralipid has been used as a tracer substance for serum TG [24, 25]. Levels of serum TG-rich lipoprotein can be determined by nephelometry [26]. In our earlier investigation, we used this method to measure LSIs in diluted 100-fold with physiological saline and found them to be significantly correlated with serum TG (r=0.94, p<0.001, n=151) [2?]. J. Clin. Biochem. Nutr.

5 FRACTIONAL REMOVAL RATE OF FAT EMULSION AND LIPOPROTEIN LIPASE 41 Reaven et al. measured endogenously labeled Sf ~2o plasma after intravenous injection of isotopically labeled glycerol and reported that removal of these TG follows the kinetics of a saturable system [28]. However, such techniques using labeled FFA or glycerol are based on assumptions, which have been criticized [29-31]. Although the fat emulsion tolerance test cannot be employed to measure the removal of endogenous TG, the K2 obtained with this test can be used to estimate the fractional catabolic rate of serum TG-rich lipoprotein. There is reportedly a good correlation between the K2 of Intralipid and the fractional catabolic rate (FCR) of VLDL-apo B [32]. Thus, the catabolism of TGrich lipoprotein can be indirectly determined from the K2 obtained with the fat emulsion tolerance test. We previously reported the high reliability and reproducibility of K2 determination by the fat emulsion tolerance test, suggesting that this test can be readily used to study the effects of diet, drug treatment, physical exercise and the like on the metabolism of serum lipids [4, 33]. In the present study, K2 discriminated normolipidemic subjects and hypertriglyceridemic subjects. However, LPL activity did not discriminate normolipidemic subjects and hypertriglyceridemic subjects. Considering that postheparin LPL mass and activity are affected by a dose of heparin, it cannot denied that postheparin LPL mass and activity in this study contain artificial factor. It is reported that maximal response is obtained at a heparin dose of 100 IU/kg [34, 35] or 75 IU/kg [36] for the release of lipase activities. It is doubted that a heparin dose of 30 IU/kg in this study is not adequate to obtain maximal response for the release of lipase. Therefore, it is speculated that K2 was not significantly correlated with the postheparin plasma LPL activity in hypertriglyceridemic subjects and that it was not possible to clearly discriminate normolipidemic subjects and hypertriglyceridemic subjects by quantifying LPL mass and activity because of a heparin dose of 30 IU/kg. In the present study, we observed a positive correlation between K2 and mass and activity in postheparin plasma in the total group of subjects. This represents the first confirmation that the kinetics obtained with an intravenously injected fat emulsion reflect LPL mass and activity in postheparin plasma. This work was supported in part by Kissei Pharmaceutical Co. Ltd. REFERENCES 1. Austin, M.A. (1991): Plasma triglyceride and coronary heart disease. Arterioscler. Thromb., 11, Sakuma, N., Hasegawa, Y., Ikeuchi, K., Cui, R., Ichikawa, T., and Fujinami, T. (1987): A simplified intravenous fat emulsion tolerance test. J. Clin. Biochem. Nutr, 3, Ikeuchi, R., Sakuma, N., Ichikawa, T., Hirata, H., Noguchi, Y., Yoshikawa, M., Hibino, T., Iwata, S., Hasegawa, Y., and Fujinami, T. (1991): Reproducibility of removal rate (K2) in intravenous fat emulsion tolerance test (II). J. Jpn. Atheroscl. Soc., 19, Ikeuchi, R., Sakuma, N., Hibino, T., Sato, T., Kamiya, Y., Kawaguchi, M., Ohte, Y., and Hayano, J. (1994): Increased serum triglyceride clearance, unchanged cholesterol ester transfer protein activity, and elevated HDL cholesterol during treatment of hypertriglyceridemia with bezafibrate. Curr. They: Res., 55, Vol. 31, 2002

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