Type I Hyperlipoproteinemia Caused by Lipoprotein Lipase Defect Lipid-Interface Recognition Was Relieved by Administration of Medium-Chain Triglyceride
in
Kohji Shirai, Junji Kobayashi, Hidekuni Inadera, Yuji Ohkubo, Seijiro Mori, Yasushi Saito, and Sho Yoshida We have previously reported lipoprotein lipase with a defect of lipid-interface recognition in a patient with type I hyperlipoproteinemia. In this patient, lipoprotein lipase from post-heparin plasma (PHP) hydrolyzed monomeric substrate tributyrin, but scarcely hydrolyzed triolein emulsified with Triton X-100 and that in very-low-density lipoproteins ([VLDL] d < 1.006 g / mL), and did not bind to VLDL. The triglyceride (TG) level of this patient did not decrease to less than 1,000 mg / dL with a low-fat diet (1,400 kcal containing 10 g fat/d). When the patient took 30 g medium-chain TG (MCT) in addition to the 1,400-kcal diet, her serum TG level decreased to 250 mg/dL and her clinical signs improved. The low clearance rate of serum TG with heparin injection improved after intake of MCT. Caproic acid levels were maintained at 1.4% and 2.6% in chylomicrons and VLDL after MCT intake, respectively. The patient’s lipoprotein lipase hydrolyzed triolein emulsified with 2% tricaprin at the same rate as that of control lipoprotein lipase. The patient’s lipoprotein lipase-catalyzed hydrolyzing rate of triolein in chylomicrons obtained after MCT administration was also enhanced up to 70% of that of control lipoprotein lipase. These findings suggest that hypertriglyceridemia caused by lipoprotein lipase with a defect in lipid-interface recognition could be relieved with the administration of medium-chain TG, and that one of the mechanisms of this effect might be a modification of TG-rich lipoproteins by MCT. Copyright 0 7992 by W.8. Saunders Company
EVERE HYPERTRIGLYCERIDEMIA is often associated with pancreatitis. I.2One cause of hypertriglyceridemia is the dysfunction of lipoprotein lipase.3-6 Recently, we reported a type I hyperlipoproteinemic patient who suffered from recurrent pancreatitis and whose lipoprotein lipase purified from post-heparin plasma (PHP) could hydrolyze tributyrin, but not triolein emulsified with Triton X-100 or that in very-low-density lipoproteins (VLDL).7 Based on the functional model of lipoprotein lipase, which is composed of the lipid-interface recognition site (IRS) and catalytic site,s.i’ one possible mechanism of our patient’s lipoprotein lipase dysfunction was abnormality of IRS. Interestingly, her lipoprotein lipase hydrolyzed triolein emulsified with lysophosphatidylcholine,’ indicating that her lipoprotein lipase might also hydrolyze triolein in certain configurations of lipid interface. The use of medium-chain triglyceride (MCT) in the treatment of hyperchylomicronemia has been reported,i2 since MCT does not form chylomicrons and is carried by the portal vein. But the precise mechanism of its absorption and the mdication of the type of hyperchylomicronemia have not yet been established. s
This
report
deals
with
the
effect
of administration
of
MCT in a type I hyperlipoproteinemic patient with possible IRS-impaired lipoprotein lipase, and with the studies of the mechanism of its effect.
activity was detectable. but hepatic lipase activity was 5.11 pmoli mL/h (range, 3.5 to 6.5) when measured with triolein emulsified with Triton X-100 as a substrate. The lipoprotein lipase mass was 470 ng/mL as measured by the method of Goers et al’? (mean + SD of 20 normal, healthy individuals, 441 ‘_ 84 ng/mL).
Properties qf Lipoprotein Lipase Purified From PHP PHP from the patient was applied to a heparin-Sepharose column and the product was eluted with 0.75 moi/L NaCl and then with 1.6 mot/L NaCI. The lipoprotein lipase fraction eluted with 1.6 mol/L NaCl showed tributyrin-hydrolyzing activity, but no triolein-hydrolyzing activity.‘However, this lipoprotein lipase fraction could hydrolyze triolein emulsified with lysophosphatidylcholine.7
Composition of Therapeutic Diets The low-fat diet (1,400 kcalid) was composed of 60 g protein. 10 g long-chain fat, and 263 g carbohydrate. The high-protein diet (1,200 kcalid) was composed of 130 g protein, 10 g fat, and 148 g carbohydrate. The MCT-containing diet (1,500 or 1,600 kcalid) was composed of 60 g protein, 20 g long-chain fat, 202 or 227 g carbohydrate, and 30 g MCT. The MCT diet was kindly supplied by Snow Brand Milk Products (Tokyo. Japan) and it contained CR0 (75%) and ClO:O (25%).
Analysis of Fatty Acid Composition Lipids in chytomicrons and VLDL were extracted with chloroform-methanol 2:l (vol/vol). The fatty acid composition of esterified lipids was determined by gas-liquid chromatography in a
SUBJECT AND METHODS
Patient Profile and Properties of Lipoprotein Lipase in Her PHP The patient was a 14-year-old girl whose clinical features have been previously reported.’ Briefly, from the age of 10 she had severe hypertriglyceridemia and suffered from pancreatitis recurrently. When she ate a low-fat diet (1,400 kcalid) containing 10 g fat. 40 g protein, and 288 g carbohydrate, her serum triglyceride (TG) level was 1,630 mg/dL (chylomicron-TG, 628 mg/dL; VLDLTG, 742 mg/dL; low-density lipoprotein [LDL]-TG, 164 mg/dL; high-density lipoprotein [HDL]-TG, 90 mg/dL) and her total cholesterol level was 248 mg/dL. In PHP, no lipoprotein lipase
Metabolism,
Vo141, No 11 (November), 1992: pp 1161-1164
From the Second Department qf Internal Medicine, School of Medicine, Chiba lInil~e&y, Chiba. Japan. Supported in part by Grant No. 02671084 from the Ministty of Education, Science and Culture sf Japan, The Mochida Memorial Foundation, and The Kashiwado Memorial Foundation. Address reprint requests to Yasushi Saito, MD, Second Department of Internal Medicine, School of Medicine, Chiba University, l-8-1, Inohana, Chiba 280, Japan. Copvright 0 1992 by W.B. Saunders Company 0026-049519214111-0003$03.00/0 1161
1162
SHIRAI ET AL
model GC7-A chromatograph method reported previously.14
(Shimadzu, Kyoto, Japan) by a
TG Clearance by Heparin Injection The patient fasted overnight, and then heparin (50 USP/kg) was injected intravenously. Blood samples were obtained at 0, 15, and 30 minutes after the injection. Plasma TG content was immediately measured by the enzyme method using an autoanalyzer. The controls were type IV hyperlipoproteinemic patients with TG levels between 180 and 300 mg/dL and normal ranges of lipoprotein lipase activities in PHP. Hydrolysis of Triolein Emulsified With Tricaprin Mixtures of 2 PCi glyceryl tri[l-14C]oleate (New England Nuclear, Boston, MA; specific activity, 50 mCi/mmol), 40 mg glyceryl trioleate (Sigma, St Louis, MO), and various amounts of tricaprin (0 to 4 mg) were evaporated under Nz gas. Then 2 mL 1 mol/L Tris hydrochloride (pH 8.4) was added, the mixtures were sonicated for 5 minutes at 0°C in a sonicator, and 2 mL 20% bovine serum albumin was added. The reaction mixture for assay of lipase activity contained 75 ~.LLof this substrate mixture, 0.5 pg human apolipoprotein C-II, and 1 U esterase (10 nmol tributyrin hydrolyzed/h) of the patient’s lipoprotein lipase or normal lipoprotein lipase purified on heparin-Sepharose in a final volume of 0.25 mL. The radioactivity of [r4C]oleic acid released was measured as described previously.’ Hydrolysis of Tri[14CJoleoyl Glycerol in Chylomicrons by Lipoprotein Lipase Chylomicrons were separated from plasma by centrifugation at 26,000 x g for 30 minutes. Plasma was obtained 3 hours after ingesting a soup containing 20 g butter, and again on the seventh day after ingestion of soup containing 20 g MCT. During that period, MCT was administered at a dose of 30 g daily. The content of medium-chain fatty acids in chylomicrons after taking MCT were 2.1% ClO:O and 3.0% C12:O. Glyceryl tri[14C]oleate glycerol incorporation into chylomicrons was achieved by the method of Fielding15 using dimetbylsulfoxide. The reaction mixture contained 130 kg TG in chylomicrons, 10% fatty acid-free bovine serum albumin, 1 U esterase of the patient’s lipoprotein lipase or normal lipoprotein lipase, and 0.1 mol/L Tris hydrochloride (pH 7.4) in a final volume of 250 JLL. The incubation was performed for 60 minutes, and the radioactivities of released [14C]oleic acid were counted as described previously.7 RESULTS
Effects of Administration of MCT-Containing Diet on Serum Lipids and Clinical Features When the patient was kept on a very-low-energy regimen of 1,000 K, Cal/d by intravenous hyperalimentation, her TG level decreased to 273 mg/dL, accompanied by a decrease in body weight. With a low-fat diet of 1,400 kcal/d, her TG level increased to 1,630 mg/dL (Fig 1). Since this low-fat diet was not successful, we tried a high-protein diet composed of 130 g protein, 10 g fat, and 125 g carbohydrate with a total energy content of 1,200 kcal/d. As shown in Fig 1, her serum TG level decreased to between 700 and 800 mg/dL, but her body weight also decreased by 1 kg over a 6-week period. Next, she was given an MCT-containing diet composed of 10 g fat (long-chain TG), 8 g medium-chain
Jan
Jan
Jan
Jan Jan Jan Mar Apr
Fig 1. Effect of low-fat diet (I.400 kc-al). high-protein diet, and MCT diet on serum lipids and body weight. Diet compositions are described in the Methods. T. Chol, total cholesterol; HDL-C, HDL cholesterol.
TG, 70 g protein, and 310 g carbohydrate (1,200 kcal/d). Her TG level decreased to 300 mg/mL. Then her total energy uptake increased to 1,500 kcal/d by the further addition of 30 g MCT. Her TG level was still between 200 and 300 mg/dL, and she gained 2.5 kg over a 3-month period. Her HDL cholesterol level increased from 18 mg/dL to 50 mg/dL, since then, pancreatitis episodes have not been seen. With the cessation of MCI administration, her TG level increased to 750 mg/dL and decreased again when MCT therapy was resumed (Fig 1). MCT was also administered to another type I hyperlipidemic patient (a 41-year-old man) whose lipoprotein lipase mass was not detected in his PHP. His serum TG level was 1,030 mg/dL when he was on a low-fat diet of 1,800 kcal/d. MCT administration (20 g/d) did not reduce his TG level below 860 mg/dL (data not shown). Lipoprotein Lipase and Hepatic Lipase Activities in PHP Measured With T&on X-100-Emulsified [24CJTriolein
Before MCT administration, hepatic lipase activity in plasma obtained 10 minutes after the injection of 10 U sodium heparinik was 5.11 (normal range, 5.01 & 1.50 krnol . mL-l . h-l), but no lipoprotein lipase activity was detectable (normal range, 2.85 t 0.85 prnol . mL-i . h-l). After MCT treatment, hepatic lipase activity was 4.05, but lipoprotein lipase activity was still not detectable.
MCT AND LIPOPROTEIN
1163
LIPASE DEFECT
Analysis of Fatty Acid Composition in Chylomicrons and VLDL During Intake of MCT
%
4 Heparin
0 Patient MCT (-) ,
(5oulkg) I.V.
TG = 710 fmg/dU
Chylomicrons and VLDL were obtained 2 hours after each meal during intake of 30 g MCTid for 2 weeks. The level of ClO:O was 1.4% and 2.6% in chylomicrons and VLDL, respectively. These values were not detected in these TG-rich lipoproteins during diet intake without MCT (Table 1).
0 Patient
YCT (6) , TG=273 l
Patient MCT (+), TG = 268
- Type IV
Effect of MCTAdministrafion on TG Clearance After Hepatin Injection
Subjects TG=;z
When TG levels were 273 mg/dL on a very-low-caloric diet and 710 mg/dL on a high-protein diet, the rate of decrease of TG levels with heparin injection was 22% and 12%, respectively. In control subjects, these values were higher than 50% (Fig 2). After the patient was on a diet containing MCT for 4 weeks, the rate of decrease in TG levels by hcparin injection increased to 40%. Effect of the Patient’s Lipoprotein Lipase on Triolein Emulsified With Tricaptin The amount of the patient’s lipoprotein lipase and that of normal controls were adjusted to the same tributyrinhydrolyzing activity (equal to 1 U esterase activity), and their effects on triolein emulsified with or without tricaprin were compared (Fig 3). The patient’s lipoprotein lipase scarcely hydrolyzed triolein without tricaprin, but with tricaprin the triolein-hydrolyzing activity increased, with the rate becoming almost the same as that of controls in the presence of only 2% tricaprin. Chylomicron Tri[14C]oleoyl Glycerol Hydrolysis by the Patient’s Lipoprotein Lipase: Comparison Between Chylomicrons With and Without Medium-Chain Fatty Acids
11 0
1
I
15
30
1
Time (min) Fig 2. Effect of MCT administration on TG clearance after heparin injection. The procedure used is described in the Methods. Tests on the patient were performed when her TG level was 273 mg/dL during the intravenous administration of nutrients (1,000 kcal/d). and 710 mg/dL with a high-protein diet (1,200 kcal/d). These levels were obtained before treatment; the TG level of 268 mg/dL was attained with MCT treatment.
300 mg/dL, from a level of 1,320 mg/mL with a low-fat diet of 1,400 kcal/d. She gained weight and no longer suffered from pancreatitis. The effectiveness of MCT was confirmed by temporarily discontinuing MCT administration: the TG level increased to 7.50 mg/dL, and it decreased again with the resumption of MCT. It is generally considered that MCT absorbed into the
mean &SD
Hydrolysis of tri[rY]oleoyl glycerol in chylomicrons by the patient’s lipoprotein lipase was 14% of that of the control (Table 2), but the hydrolysis of tri[*jC]oleoyl glycerol in chylomicrons obtained after MCT administration was enhanced to 60% of that of the control. DISCUSSION
When the patient had 30 g MCT daily added to a diet of 1,600 kcalid, her TG level decreased to between 200 and Table 1. Effect of MCT Administration
on Fatty Acid Composition of
Chylomicrons and VLDL Before Administration Fatty Acid
Chylomicrons
After Administration
VLDL
ClO:O
c12:o c14:o C16:O
1.7
1.5
Chylomicrons
VLDL
1.4
2.6
3.0
4.0
4.3
3.7
8.5
2.7
22.5
24.3
22.4
29.0
C16:l
9.8
9.8
10.8
12.0
C18:O
8.6
8.7
4.9
5.2
C18:l
28.6
28.7
24.3
27.0
C18:2
17.0
17.0
18.6
15.3
NOTE. Values are percentages.
Added Tricaprin Fig 3. Effect of addition of tricaprin on hydrolysis of triolein by the patient’s lipoprotein lipase. Preparation of substrate emulsions and assay conditions are described in the Methods. Values were derived from studies performed in triplicate.
1164
SHIRAI ET AL
Table 2. Hydrolysis of Chylomicron-Triolein
by Patient’s Lipoprotein
Lipase: Effect of MCT Administration Chylomicron
Tri[“C]oleoyl
Without MCT Diet
Glycerol Hydrolysis* With MCT Diet
Lipoprotein lipase Patient
0.3 k 0.1
3.1 -t 0.2
Control
4.4 2 0.2
4.6 -c 0.2
NOTE. Values are means f SD of the study done in triplicate. *Released[14C]oleic acid in nmol/U esterase/h.
intestine does not form chylomicrons and is transferred to the liver via the portal vein, 12~16 and is then metabolized into acetyl coenzyme A. Based on this metabolic pathway, MCT is thought to be effective for type I patients because it does not form chylomicrons and it behaves like carbohydrates. Our patient’s hypertriglyceridemia could not be relieved with a low-fat diet of 1,400 kcal/d. If MCT is metabolized like carbohydrates, her TG level should not have decreased below the level obtained with a low-fat diet. Therefore, some other mechanisms appear to be involved. Heparin injection studies (Fig 1) showed that her low rate of TG clearance became enhanced after beginning an MCT-containing diet. This enhancement might be due to the increment of lipoprotein lipase enzyme mass and/or increased susceptibility of VLDL or chylomicron-TG to lipoprotein lipase; we studied the latter possibility. Fatty acid analysis showed that capric acid was present in 1.7% or 3.0% of all fatty acids in her chylomicrons or VLDL (Table 1). These results suggested that parts of medium-chain fatty acids were incorporated into chylomicrons in the intestine and also into VLDL in the liver, and the properties of these
lipoproteins might become modified, enabling triolein to be hydrolyzed by the patient’s lipoprotein lipase. Therefore, we tested whether the patient’s abnormal lipoprotein lipase could hydrolyze triolein when the substrate was modified with tricaprin in vitro (Fig 3); the patient’s lipoprotein lipase did indeed hydrolyze triolein emulsified with tricaprin. This enhancement was observed with only 1% to 5% tricaprin, which is comparable to the amount in chylomicrons or VLDL, when the patient followed a diet containing MCT. Compared with the hydrolyzing rate of chylomicron triolein obtained without MCT treatment, the rate after MCT intake was considerably enhanced (Table 2). These results suggest that capric acid might change the surface of chylomicrons so that the patient’s lipoprotein lipase recognizes and hydrolyzes triolein. These results strongly suggest that the improved TG clearance rate with MCT administered as shown in Fig 2 might be mainly due to increased susceptibility of chylomicrons or VLDL-TG to the patient’s lipoprotein lipase, even though other mechanisms might be involved. The precise mechanism by which MCT modifies TG-rich lipoproteins so that a patient’s lipoprotein lipase hydrolyzes triolein is still not fully clarified. However, it can be concluded that MCT administration may improve some cases of type I hyperlipoproteinemia in patients whose lipoprotein lipase has a defect in lipid-interface recognition of TG-rich lipoproteins. ACKNOWLEDGMENT
We are very grateful to Dr Michael C. Schotz for the measurement of lipoprotein lipase mass.
REFERENCES
1. Brunzell JD: Familial lipoprotein lipase deficiency and other causes of the chylomicronemia syndrome, in Striver CR, Beaudet AL, Sly WS, et a! (eds): The Metabolic Basis of Inherited Disease. New York, NY, McGraw-Hill, 1989, pp 11651180 2. Fredrickson DS, Levy RI: Familial hyperlipoproteinemia, in Stanbury JB, Wyngaarden JB, Fredrickson DS (eds): Metabolic Basis of Inherited Disease (ed 3). New York, NY, McGraw-Hill, 1972, pp X5-614 3. Have1 RJ: Approach to the patient with hyperlipidemia. Med Clin North Am 66:319-334.1982 4. Krauss RM, Levy RI, Fredrickson D: Selective measurement of two lipase activities in post-heparin plasma from normal subjects and patients with hyperlipoproteinemia. J Clin Invest 54:11071124,1974 5. Olivercrona T, Chernick SS, Olivercrona GB, et a!: Combined lipase deficiency in mice. J Bio! Chem 260:2552-2557C, 1985 6. Karen L, Kirchgessner TG, Lusis AJ, et al: Human lipoprotein lipase complementary DNA sequence. Science 235:1638-1641, 1987 7. Kobayashi J, Shirai K, Saito Y, et a!: Lipoprotein lipase with a defect in lipid interface recognition in a case of type I hyperlipidemia. Eur J Clin Invest 19:424-432, 1989 8. Shirai K, Matsuoka N, Jackson RL: Interaction of lipoprotein
lipase with phospholipid vesicles: Role of apolipoprotein CII and heparin. Biochim Biophys Acta 665:504-510, 1981 9. Shirai K, Jackson RL: Lipoprotein lipase catalyzed hydrolysis ofp-nitrophenyl butyrate. J Biol Chem 257:1253-1258,1982 10. Bengtsson G, Olivecrona T: Lipoprotein lipase: Modification of its kinetic properties by mild tryptic digestion. Eur J Biochem 113:547-554,198l 11. Rapp D, Olivecrona T: Kinetics of milk lipoprotein lipase. Studies with tributyrin. Eur J Biochem 91:379-385, 1978 12. Greenberger NJ, Skillman TG: Medical progress, medium chain triglyceride, physiologic considerations and clinical implication. N Engl J Med 280:1045-1058, 1969 13. Goers JWF, Pedersen ME, Kern PA, et al: An enzymelinked immunoassay for lipoprotein lipase. Anal Biochem 66:27-35, 1989 14. Morisaki N, Kanzaki T, Fujiyama Y, et al: Metabolism of n-3 polyunsaturated fatty acids and modification of phospholipids in cultured rabbit smooth muscle cells. J Lipid Res 26:930-939,1985 15. Fielding CJ: Validation of a procedure for exogenous isotopic labeling of lipoprotein triglyceride with radioactive triolein. Biochim Biophys Acta 573:255-265,198O 16. Hashim SA, Berger SS Jr, Krell K, et al: Intestinal absorption and mode of transport in portal vein of medium chain fatty acids. J Clin Invest 43:1238, 1964
in
Kohji Shirai, Junji Kobayashi, Hidekuni Inadera, Yuji Ohkubo, Seijiro Mori, Yasushi Saito, and Sho Yoshida We have previously reported lipoprotein lipase with a defect of lipid-interface recognition in a patient with type I hyperlipoproteinemia. In this patient, lipoprotein lipase from post-heparin plasma (PHP) hydrolyzed monomeric substrate tributyrin, but scarcely hydrolyzed triolein emulsified with Triton X-100 and that in very-low-density lipoproteins ([VLDL] d < 1.006 g / mL), and did not bind to VLDL. The triglyceride (TG) level of this patient did not decrease to less than 1,000 mg / dL with a low-fat diet (1,400 kcal containing 10 g fat/d). When the patient took 30 g medium-chain TG (MCT) in addition to the 1,400-kcal diet, her serum TG level decreased to 250 mg/dL and her clinical signs improved. The low clearance rate of serum TG with heparin injection improved after intake of MCT. Caproic acid levels were maintained at 1.4% and 2.6% in chylomicrons and VLDL after MCT intake, respectively. The patient’s lipoprotein lipase hydrolyzed triolein emulsified with 2% tricaprin at the same rate as that of control lipoprotein lipase. The patient’s lipoprotein lipase-catalyzed hydrolyzing rate of triolein in chylomicrons obtained after MCT administration was also enhanced up to 70% of that of control lipoprotein lipase. These findings suggest that hypertriglyceridemia caused by lipoprotein lipase with a defect in lipid-interface recognition could be relieved with the administration of medium-chain TG, and that one of the mechanisms of this effect might be a modification of TG-rich lipoproteins by MCT. Copyright 0 7992 by W.8. Saunders Company
EVERE HYPERTRIGLYCERIDEMIA is often associated with pancreatitis. I.2One cause of hypertriglyceridemia is the dysfunction of lipoprotein lipase.3-6 Recently, we reported a type I hyperlipoproteinemic patient who suffered from recurrent pancreatitis and whose lipoprotein lipase purified from post-heparin plasma (PHP) could hydrolyze tributyrin, but not triolein emulsified with Triton X-100 or that in very-low-density lipoproteins (VLDL).7 Based on the functional model of lipoprotein lipase, which is composed of the lipid-interface recognition site (IRS) and catalytic site,s.i’ one possible mechanism of our patient’s lipoprotein lipase dysfunction was abnormality of IRS. Interestingly, her lipoprotein lipase hydrolyzed triolein emulsified with lysophosphatidylcholine,’ indicating that her lipoprotein lipase might also hydrolyze triolein in certain configurations of lipid interface. The use of medium-chain triglyceride (MCT) in the treatment of hyperchylomicronemia has been reported,i2 since MCT does not form chylomicrons and is carried by the portal vein. But the precise mechanism of its absorption and the mdication of the type of hyperchylomicronemia have not yet been established. s
This
report
deals
with
the
effect
of administration
of
MCT in a type I hyperlipoproteinemic patient with possible IRS-impaired lipoprotein lipase, and with the studies of the mechanism of its effect.
activity was detectable. but hepatic lipase activity was 5.11 pmoli mL/h (range, 3.5 to 6.5) when measured with triolein emulsified with Triton X-100 as a substrate. The lipoprotein lipase mass was 470 ng/mL as measured by the method of Goers et al’? (mean + SD of 20 normal, healthy individuals, 441 ‘_ 84 ng/mL).
Properties qf Lipoprotein Lipase Purified From PHP PHP from the patient was applied to a heparin-Sepharose column and the product was eluted with 0.75 moi/L NaCl and then with 1.6 mot/L NaCI. The lipoprotein lipase fraction eluted with 1.6 mol/L NaCl showed tributyrin-hydrolyzing activity, but no triolein-hydrolyzing activity.‘However, this lipoprotein lipase fraction could hydrolyze triolein emulsified with lysophosphatidylcholine.7
Composition of Therapeutic Diets The low-fat diet (1,400 kcalid) was composed of 60 g protein. 10 g long-chain fat, and 263 g carbohydrate. The high-protein diet (1,200 kcalid) was composed of 130 g protein, 10 g fat, and 148 g carbohydrate. The MCT-containing diet (1,500 or 1,600 kcalid) was composed of 60 g protein, 20 g long-chain fat, 202 or 227 g carbohydrate, and 30 g MCT. The MCT diet was kindly supplied by Snow Brand Milk Products (Tokyo. Japan) and it contained CR0 (75%) and ClO:O (25%).
Analysis of Fatty Acid Composition Lipids in chytomicrons and VLDL were extracted with chloroform-methanol 2:l (vol/vol). The fatty acid composition of esterified lipids was determined by gas-liquid chromatography in a
SUBJECT AND METHODS
Patient Profile and Properties of Lipoprotein Lipase in Her PHP The patient was a 14-year-old girl whose clinical features have been previously reported.’ Briefly, from the age of 10 she had severe hypertriglyceridemia and suffered from pancreatitis recurrently. When she ate a low-fat diet (1,400 kcalid) containing 10 g fat. 40 g protein, and 288 g carbohydrate, her serum triglyceride (TG) level was 1,630 mg/dL (chylomicron-TG, 628 mg/dL; VLDLTG, 742 mg/dL; low-density lipoprotein [LDL]-TG, 164 mg/dL; high-density lipoprotein [HDL]-TG, 90 mg/dL) and her total cholesterol level was 248 mg/dL. In PHP, no lipoprotein lipase
Metabolism,
Vo141, No 11 (November), 1992: pp 1161-1164
From the Second Department qf Internal Medicine, School of Medicine, Chiba lInil~e&y, Chiba. Japan. Supported in part by Grant No. 02671084 from the Ministty of Education, Science and Culture sf Japan, The Mochida Memorial Foundation, and The Kashiwado Memorial Foundation. Address reprint requests to Yasushi Saito, MD, Second Department of Internal Medicine, School of Medicine, Chiba University, l-8-1, Inohana, Chiba 280, Japan. Copvright 0 1992 by W.B. Saunders Company 0026-049519214111-0003$03.00/0 1161
1162
SHIRAI ET AL
model GC7-A chromatograph method reported previously.14
(Shimadzu, Kyoto, Japan) by a
TG Clearance by Heparin Injection The patient fasted overnight, and then heparin (50 USP/kg) was injected intravenously. Blood samples were obtained at 0, 15, and 30 minutes after the injection. Plasma TG content was immediately measured by the enzyme method using an autoanalyzer. The controls were type IV hyperlipoproteinemic patients with TG levels between 180 and 300 mg/dL and normal ranges of lipoprotein lipase activities in PHP. Hydrolysis of Triolein Emulsified With Tricaprin Mixtures of 2 PCi glyceryl tri[l-14C]oleate (New England Nuclear, Boston, MA; specific activity, 50 mCi/mmol), 40 mg glyceryl trioleate (Sigma, St Louis, MO), and various amounts of tricaprin (0 to 4 mg) were evaporated under Nz gas. Then 2 mL 1 mol/L Tris hydrochloride (pH 8.4) was added, the mixtures were sonicated for 5 minutes at 0°C in a sonicator, and 2 mL 20% bovine serum albumin was added. The reaction mixture for assay of lipase activity contained 75 ~.LLof this substrate mixture, 0.5 pg human apolipoprotein C-II, and 1 U esterase (10 nmol tributyrin hydrolyzed/h) of the patient’s lipoprotein lipase or normal lipoprotein lipase purified on heparin-Sepharose in a final volume of 0.25 mL. The radioactivity of [r4C]oleic acid released was measured as described previously.’ Hydrolysis of Tri[14CJoleoyl Glycerol in Chylomicrons by Lipoprotein Lipase Chylomicrons were separated from plasma by centrifugation at 26,000 x g for 30 minutes. Plasma was obtained 3 hours after ingesting a soup containing 20 g butter, and again on the seventh day after ingestion of soup containing 20 g MCT. During that period, MCT was administered at a dose of 30 g daily. The content of medium-chain fatty acids in chylomicrons after taking MCT were 2.1% ClO:O and 3.0% C12:O. Glyceryl tri[14C]oleate glycerol incorporation into chylomicrons was achieved by the method of Fielding15 using dimetbylsulfoxide. The reaction mixture contained 130 kg TG in chylomicrons, 10% fatty acid-free bovine serum albumin, 1 U esterase of the patient’s lipoprotein lipase or normal lipoprotein lipase, and 0.1 mol/L Tris hydrochloride (pH 7.4) in a final volume of 250 JLL. The incubation was performed for 60 minutes, and the radioactivities of released [14C]oleic acid were counted as described previously.7 RESULTS
Effects of Administration of MCT-Containing Diet on Serum Lipids and Clinical Features When the patient was kept on a very-low-energy regimen of 1,000 K, Cal/d by intravenous hyperalimentation, her TG level decreased to 273 mg/dL, accompanied by a decrease in body weight. With a low-fat diet of 1,400 kcal/d, her TG level increased to 1,630 mg/dL (Fig 1). Since this low-fat diet was not successful, we tried a high-protein diet composed of 130 g protein, 10 g fat, and 125 g carbohydrate with a total energy content of 1,200 kcal/d. As shown in Fig 1, her serum TG level decreased to between 700 and 800 mg/dL, but her body weight also decreased by 1 kg over a 6-week period. Next, she was given an MCT-containing diet composed of 10 g fat (long-chain TG), 8 g medium-chain
Jan
Jan
Jan
Jan Jan Jan Mar Apr
Fig 1. Effect of low-fat diet (I.400 kc-al). high-protein diet, and MCT diet on serum lipids and body weight. Diet compositions are described in the Methods. T. Chol, total cholesterol; HDL-C, HDL cholesterol.
TG, 70 g protein, and 310 g carbohydrate (1,200 kcal/d). Her TG level decreased to 300 mg/mL. Then her total energy uptake increased to 1,500 kcal/d by the further addition of 30 g MCT. Her TG level was still between 200 and 300 mg/dL, and she gained 2.5 kg over a 3-month period. Her HDL cholesterol level increased from 18 mg/dL to 50 mg/dL, since then, pancreatitis episodes have not been seen. With the cessation of MCI administration, her TG level increased to 750 mg/dL and decreased again when MCT therapy was resumed (Fig 1). MCT was also administered to another type I hyperlipidemic patient (a 41-year-old man) whose lipoprotein lipase mass was not detected in his PHP. His serum TG level was 1,030 mg/dL when he was on a low-fat diet of 1,800 kcal/d. MCT administration (20 g/d) did not reduce his TG level below 860 mg/dL (data not shown). Lipoprotein Lipase and Hepatic Lipase Activities in PHP Measured With T&on X-100-Emulsified [24CJTriolein
Before MCT administration, hepatic lipase activity in plasma obtained 10 minutes after the injection of 10 U sodium heparinik was 5.11 (normal range, 5.01 & 1.50 krnol . mL-l . h-l), but no lipoprotein lipase activity was detectable (normal range, 2.85 t 0.85 prnol . mL-i . h-l). After MCT treatment, hepatic lipase activity was 4.05, but lipoprotein lipase activity was still not detectable.
MCT AND LIPOPROTEIN
1163
LIPASE DEFECT
Analysis of Fatty Acid Composition in Chylomicrons and VLDL During Intake of MCT
%
4 Heparin
0 Patient MCT (-) ,
(5oulkg) I.V.
TG = 710 fmg/dU
Chylomicrons and VLDL were obtained 2 hours after each meal during intake of 30 g MCTid for 2 weeks. The level of ClO:O was 1.4% and 2.6% in chylomicrons and VLDL, respectively. These values were not detected in these TG-rich lipoproteins during diet intake without MCT (Table 1).
0 Patient
YCT (6) , TG=273 l
Patient MCT (+), TG = 268
- Type IV
Effect of MCTAdministrafion on TG Clearance After Hepatin Injection
Subjects TG=;z
When TG levels were 273 mg/dL on a very-low-caloric diet and 710 mg/dL on a high-protein diet, the rate of decrease of TG levels with heparin injection was 22% and 12%, respectively. In control subjects, these values were higher than 50% (Fig 2). After the patient was on a diet containing MCT for 4 weeks, the rate of decrease in TG levels by hcparin injection increased to 40%. Effect of the Patient’s Lipoprotein Lipase on Triolein Emulsified With Tricaptin The amount of the patient’s lipoprotein lipase and that of normal controls were adjusted to the same tributyrinhydrolyzing activity (equal to 1 U esterase activity), and their effects on triolein emulsified with or without tricaprin were compared (Fig 3). The patient’s lipoprotein lipase scarcely hydrolyzed triolein without tricaprin, but with tricaprin the triolein-hydrolyzing activity increased, with the rate becoming almost the same as that of controls in the presence of only 2% tricaprin. Chylomicron Tri[14C]oleoyl Glycerol Hydrolysis by the Patient’s Lipoprotein Lipase: Comparison Between Chylomicrons With and Without Medium-Chain Fatty Acids
11 0
1
I
15
30
1
Time (min) Fig 2. Effect of MCT administration on TG clearance after heparin injection. The procedure used is described in the Methods. Tests on the patient were performed when her TG level was 273 mg/dL during the intravenous administration of nutrients (1,000 kcal/d). and 710 mg/dL with a high-protein diet (1,200 kcal/d). These levels were obtained before treatment; the TG level of 268 mg/dL was attained with MCT treatment.
300 mg/dL, from a level of 1,320 mg/mL with a low-fat diet of 1,400 kcal/d. She gained weight and no longer suffered from pancreatitis. The effectiveness of MCT was confirmed by temporarily discontinuing MCT administration: the TG level increased to 7.50 mg/dL, and it decreased again with the resumption of MCT. It is generally considered that MCT absorbed into the
mean &SD
Hydrolysis of tri[rY]oleoyl glycerol in chylomicrons by the patient’s lipoprotein lipase was 14% of that of the control (Table 2), but the hydrolysis of tri[*jC]oleoyl glycerol in chylomicrons obtained after MCT administration was enhanced to 60% of that of the control. DISCUSSION
When the patient had 30 g MCT daily added to a diet of 1,600 kcalid, her TG level decreased to between 200 and Table 1. Effect of MCT Administration
on Fatty Acid Composition of
Chylomicrons and VLDL Before Administration Fatty Acid
Chylomicrons
After Administration
VLDL
ClO:O
c12:o c14:o C16:O
1.7
1.5
Chylomicrons
VLDL
1.4
2.6
3.0
4.0
4.3
3.7
8.5
2.7
22.5
24.3
22.4
29.0
C16:l
9.8
9.8
10.8
12.0
C18:O
8.6
8.7
4.9
5.2
C18:l
28.6
28.7
24.3
27.0
C18:2
17.0
17.0
18.6
15.3
NOTE. Values are percentages.
Added Tricaprin Fig 3. Effect of addition of tricaprin on hydrolysis of triolein by the patient’s lipoprotein lipase. Preparation of substrate emulsions and assay conditions are described in the Methods. Values were derived from studies performed in triplicate.
1164
SHIRAI ET AL
Table 2. Hydrolysis of Chylomicron-Triolein
by Patient’s Lipoprotein
Lipase: Effect of MCT Administration Chylomicron
Tri[“C]oleoyl
Without MCT Diet
Glycerol Hydrolysis* With MCT Diet
Lipoprotein lipase Patient
0.3 k 0.1
3.1 -t 0.2
Control
4.4 2 0.2
4.6 -c 0.2
NOTE. Values are means f SD of the study done in triplicate. *Released[14C]oleic acid in nmol/U esterase/h.
intestine does not form chylomicrons and is transferred to the liver via the portal vein, 12~16 and is then metabolized into acetyl coenzyme A. Based on this metabolic pathway, MCT is thought to be effective for type I patients because it does not form chylomicrons and it behaves like carbohydrates. Our patient’s hypertriglyceridemia could not be relieved with a low-fat diet of 1,400 kcal/d. If MCT is metabolized like carbohydrates, her TG level should not have decreased below the level obtained with a low-fat diet. Therefore, some other mechanisms appear to be involved. Heparin injection studies (Fig 1) showed that her low rate of TG clearance became enhanced after beginning an MCT-containing diet. This enhancement might be due to the increment of lipoprotein lipase enzyme mass and/or increased susceptibility of VLDL or chylomicron-TG to lipoprotein lipase; we studied the latter possibility. Fatty acid analysis showed that capric acid was present in 1.7% or 3.0% of all fatty acids in her chylomicrons or VLDL (Table 1). These results suggested that parts of medium-chain fatty acids were incorporated into chylomicrons in the intestine and also into VLDL in the liver, and the properties of these
lipoproteins might become modified, enabling triolein to be hydrolyzed by the patient’s lipoprotein lipase. Therefore, we tested whether the patient’s abnormal lipoprotein lipase could hydrolyze triolein when the substrate was modified with tricaprin in vitro (Fig 3); the patient’s lipoprotein lipase did indeed hydrolyze triolein emulsified with tricaprin. This enhancement was observed with only 1% to 5% tricaprin, which is comparable to the amount in chylomicrons or VLDL, when the patient followed a diet containing MCT. Compared with the hydrolyzing rate of chylomicron triolein obtained without MCT treatment, the rate after MCT intake was considerably enhanced (Table 2). These results suggest that capric acid might change the surface of chylomicrons so that the patient’s lipoprotein lipase recognizes and hydrolyzes triolein. These results strongly suggest that the improved TG clearance rate with MCT administered as shown in Fig 2 might be mainly due to increased susceptibility of chylomicrons or VLDL-TG to the patient’s lipoprotein lipase, even though other mechanisms might be involved. The precise mechanism by which MCT modifies TG-rich lipoproteins so that a patient’s lipoprotein lipase hydrolyzes triolein is still not fully clarified. However, it can be concluded that MCT administration may improve some cases of type I hyperlipoproteinemia in patients whose lipoprotein lipase has a defect in lipid-interface recognition of TG-rich lipoproteins. ACKNOWLEDGMENT
We are very grateful to Dr Michael C. Schotz for the measurement of lipoprotein lipase mass.
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