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Intestinal absorption and plasma transport of dietary triglyceride and phosphatidylcholine in the carp (Cyprinus carpio)
Camp. Biochem. Physiol. Vol. 96A, No. I, pp. 45-55, Printedin Great Britain
0300-9629/90 $3.00 + 0.00 0 1990 PergamonPressplc
1990
INTESTINAL ABSORPTION AND PLASMA TRANSPORT OF DIETARY TRIGLYCERIDE AND PHOSPHATIDYLCHOLINE IN THE CARP (CYPRINUS CARPI0) NORIAKI IIJIMA,* SATOSHI AIDA,~
MITSUMASA MANKURA~
MITSU KAYAMA*
*Faculty of Applied Biological
Science, Hiroshima University, Shitami, Saijo-cho, Higashi-Hiroshima 724, Japan. Telephone: 0824-22-7 I 11; YHiroshima Prefectural Fisheries Experimental Station, Ondo-cho, Akigun, 737-12, Japan. Telephone: 0823-51-2171; and $Ikeda Tohka Industries Co. Ltd, Fukuyama 720, Japan. Telephone: 0849-54-5733 (Received
10 October 1989)
Abstract-l.
Intraluminal TG hydrolase activity of carp very slowly increased until 24 hr after dosing soybean oil. 2. In oiuo metabolic fates of dietary TG and phosphatidylcholine (PC) were investigated in carp, by measuring radioactivities of plasma lipids and lipoproteins after force-feeding [I-‘%]triolein or [l-‘4C]dioleyl PC. 3. After dosing [I-%]triolein, radioactivity was mainly distributed in carp plasma HDL and LDL fractions as TG. 4. When [l-‘4C]dioleyl PC was fed, a part of radioactivity was found both in carp plasma VLDL and LDL fractions; however, the major radioactivity distributed in HDL fraction as TG. At 20 hr after dosing, radioactivity in l-position of plasma PC was more than twice that in 2-position.
INTRODUffION
cation in blood plasma (Robinson and Mead, 1973; Kayama and Iijima, 1976; Patton et al., 1978); however, a major part of FFA are reconstituted into TG and then released as CHY and VLDL-like particles (Sire et al., 1981). Recently, we postulated that in carp, TG hydrolytic products were secreted as HDL-; LDL- and VLDL-like particles (mainly HDL) into the blood circular system after resynthesizing TG and PL in the intestinal mucosa (Iijima and Zama, 1979; Iijima et al., 1983; Iijima et al., 1985; Mankura et al., 1987; Iijima, Aida and Kayama, unpublished data). Several investigations have been made on the absorption of TG in fish; however, there is now some controversy over the above findings. Moreover, the mechanisms of digestion, absorption and transport of dietary PC have not yet been studied in fish. The present study was performed to examine the metabolic fate of dietary TG and PC in carp by force-feeding [ l-‘4C]dioleyl PC, collecting blood via the tube cannulated into dorsal aorta, and assaying the radioactivities of plasma lipids and lipoproteins.
In mammals, the mechanisms of digestion and absorption of TG have been studied in great detail. Dietary TG, partially hydrolysed to FFA and MG by pancreatic lipase in the intestinal lumen, are absorbed in the intestinal mucosa. In the intestinal mucosa, FFAs and MGs are mainly reconstituted into TG and PL which are associated with apoproteins, and released as chylomocron (CHY) and (VLDL) in the lymphatic system (Green and Glickman, 1981). On the other hand, dietary PL, especially PC, are hydrolysed to FFA and I-acyl 1ysoPC by pancreatic phospholipase A, activated by protease in the intestinal lumen (Carey et al., 1983; Arnesjo et al., 1969). FFA absorbed in the intestinal mucosa are metabolized as described above. However, several routes have been proposed about the metabolism of 1ysoPC in the intestinal mucosa: (a) re-esterification with FFA to form PC which can be utilized by CHY formation or for the constituent of intracellular membranes (Scow ef al., 1967; Mansbach, 1977); (b) complete hydrolysis and use of the released FFA of 1ysoPC for TG synthesis (Baxter, 1966; Ockner et al., 1969); and (c) direct absorption of 1ysoPC into the portal circulation (Boucrot, 1972). In fishes, several differences have been proposed for the digestion, absorption and transport of dietary TG compared with that in mammals: (a) some marine species completely hydrolysed dietary TG to FFA and glycerols (Patton et al., 1975; Leger, 1985), but in rainbow trout, it was partially hydrolysed to FFA, MG and DG in the intestinal lumen (Tocher and Sargent, 1984); and (b) part of FFA absorbed in the intestinal mucosa is directly released without esterifi-
MATERIALS AND METHODS
Isotope
L-a-[l-‘4C]dioleoyl phosphatidylcholine([l-‘4C]dioleyl PC, 114.0 mCi/mmol) and Il-14Cltriolein (103.5 mCiimmo1) were purchased from New &gland Nuclear througd the Nippon Isotope Association. NCS-solubilizer was purchased from Amersham Inc. All other chemicals were of the guaranteed grade available from commercial sources.
Carp (Cyprinus carpio), of 500-600 g body weight, were maintained in a large outdoor tank with pellet No. 5P for 45
NORIAKIIIJIMA
46
carp (Nippon Haigoshiryo December.
Co. Ltd) from November to
Intraluminal TG hydrolysis
Carp were starved for 2 days and then administered soybean oil (0.5 ml/100 g body weight) under anesthesia. After swimming freely in a tank, carp were killed at 0, 3, 6, 9, 12 and 24 hr, and the intestinal tracts were removed. The intestinal contents were squeezed and washed with physiological saline solution in an ice-cold centrifuge tube, and centrifuged at 27,000g for 20min. After removal of the upper lipid layer, the infranatant was used as the crude enzyme extract. 0.5ml of the crude enzyme extract at various intervals was incubated at 0.25 PCi of [I-i4C]triolein dissolved in 1 mg non-labelled triolein. The enzyme reaction was stopped by the addition of three drops of 3 N HCl, and then the lipids were extracted with diethyl ether according to the modified method of Patton et al. (1975). The residual lipids, after being removed from the solvent under reduced pressure. were separated by a TLC plate coated with silica
100 I-
et al.
gel 6OG (Merck), which was first developed with diethyl ether up to 2cm from origin, followed by the solvent system of benzene-chloroform-formic acid (70: 30: 2, v/v/v). The lipid bands were visualized with 0.01% (w/v) I-hydroxy-l,3,6-pyrenetrisulfonic acid t&odium salt (Kodak)-methanol under ultraviolet light, and identified with authentic lipid standards (monoolein, diolein, triolein and oleic acid). The separated lipid bands were scraped directly into the scintillation vials, and suspended in IO ml of toluene-based scintillation cocktail (0.2 g of 1,4&s[2-(5phenyloxazolyl)]-benzene and 4 g of 2,5-diphenyloxazole per 1000 ml of toluene) and 0.2 g of Cab-0-Sil (thixotropic gel powder, Packard). The radioactivity was measured with a liquid scintillation spectrometer (LSC-903, Aloka Co. Ltd). In vivo experiment The operation of dorsal aorta cannulation and force-feeding with labelled compounds were described elsewhere (Kayama and Iijima, 1976; Kayama and Tsuchiya, 1959). Each cannulated carp was force-fed 20 PCi of [1-‘4C]dioleoyl PC or 50 FCi of [l-‘4C]triolein, dissolved in 0.5 ml of carp body lipids, after anesthetization with 0.01% MS222 (ethyl m-aminobenzoate methane sulfonate, Nakarai Tesque). Then the fish was transferred into each aquarium in a constant water temperature (20°C). Blood (0.5 ml) was collected from the cannulated tube at intervals of 0, 3, 6, 9, 12, 28, 48 and 72 hr after dosing of labelled compounds. Plasma was obtained by centrifugation at l5OOg for IOmin andNaN, was added at a final concentration of 0.01%. Analysis of plasma lipids and lipoproteins
Tim
(h)
Fig. 1. In vitro TG digestion in the intestinal lumen of carp in winter. The intestinal fluid obtained at various intervals after force-feeding soybean oil was incubated for 30 min at 20°C with [l-‘4C]triolein. Then the radioactivities of MG (--n--), DG (-A-), FFA (--0 --) and TG (-a-) were determined and expressed as mole percentage as described in Materials and Methods.
Each 25 ~1 of plasma at various intervals was mixed with 150~1 of NCA-solubilizer in a scintillation vial, and then IO ml of scintillation cocktail (ACS-II, Amersham) was added after standing for 30min at room temperature. Plasma lipids were extracted with chloroform-methanol by the modified method of Folch et al. (1957). Plasma lipid classes were separated by TLC. The TLC plate was developed with the two step solvent systems of diethyl ether and n -hexane-diethyl ether-acetic acid (85 : I5 : 1, v/v/v), respectively, as described above. Phospholipids were also separated with TLC coated with kiesel gel HF containing 0.01% Na,CO, . The plate was developed with chloroformmethanol-acetic acid-H,0 (25: I5 : 4: 2, v/v/v/v). The separated lipid bands were scraped directly into the scintillation t-1 (VLDL) III(LDLl1
IV
II(LDL2) I(HDL)
Tlmfz(h)
Fig. 2. Time course changes of radioactivity distribution in carp plasma lipoprotein bands by PAGE after force-feeding [I-‘4C]triolein. Whole carp plasma at various intervals was separated by PAGE into four lipoprotein bands: Band I, HDL (-_O-); Band II, LDL, (--0-k Band III, LDL, (--A--); and Band IV, VLDL (-A-], The radioactivities of their bands and whole plasma (-•--) were counted as described in Materials and Methods.
Absorption and transport of TG and PC vials, and the radioactivity was measured with a liquid scintillation spectrometer. The scintillation cocktail for non-polar and polar lipids was toluene-based scintillator, containing Cab-O-%1 and ACS-II, respectively. The radioactivities of carp plasma lipoproteins at various intervals were analysed by polyacrylamide gel electrophoresis (PAGE) (Kayama and Iijima, 1976; Mankura et al., 1987) or high performance gel-filtration chromatography (HPGC) (Hara and Okazaki, 1986). Each plasma (50 ~1) was separated into four lipoprotein bands: Band I, Band II, Band III and Band IV, by PAGE. Moreover, each plasma (40 ~1) was loaded onto the high performance liquid chromatograph (HLC 803D, Toso Co. Ltd.), equipped with the combination of TSK gel G4OOOPW(7.5 x 600 mm, Toso Co. Ltd) and TSK gel G5OOOPW(7.5 x 600 mm, Toso Co. Ltd) protected with a guard column (TSK gel PWH), and eluted with 0.15 M NaCl at a flow rate of 0.5 ml/min, by monitoring at 280nm with a spectrophotometer (UV-8 Model II, Toso Co. Ltd). The eluent directly collected into scintillation vials at 2 min intervals was suspended in 10 ml of ACS-II, and the radioactivity was measured. Analysisof tissue lipids The carp were killed after the final blood sampling at 72 hr, and then the intestinal content and various tissues were excised. Plasma lipids were extracted with chloroform-methanol. Remaining blood corpuscles were washed three times with ice-cold physiological saline solution and lyophilized. Then, the lipids of lyophilized blood corpuscles, intestinal contents and various tissues were extracted with
47
chloroform-methanol according to the method of Bligh and Dyer (1959). Total lipids were fractionated into non-polar and polar lipids by silicic acid column chromatography (Kayama and Tsuchiya, 1965), and non-polar and polar lipids were separated by TLC with a solvent system of petroleum ether-diethyl ether-acetic acid (85 : 15: 1, v/v/v) and chloroforn-methanol-acetic acid-H,0 (25 : 15 : 4 : 2, v/v/v/v), respectively. Then, the radioactivities of various lipids bands were measured, as described above. RESULTS
Intraluminal TG hydrolysis Figure 1 shows the time course changes of TG hydrolase activity by carp intestinal fluid after forcefeeding with soybean oil. TG hydrolase activity was very low until 9 hr after force-feeding with soybean oil, and then gradually increased up to 24 hr. Even at 24 hr after feeding, only 25% of TG was hydrolysed, and its hydrolytic products were MG, DG and FFA. Incorporation of [l-‘4C]triolein into plasma lipidr and lipoproteins Time course changes of radioactivity distribution in carp plasma lipoprotein bands by PAGE after
force-feeding [l-‘4C]triolein are shown in Fig. 2. Almost all of the radioactivity was found in Band I;
Time (h) Fig. 3. Time course changes of radioactivity distribution in the lipid classes of carp plasma after force-feeding [l-‘4C]triolein. Carp plasma lipids at various intervals were separated into CE and WE (--a--), TG (-_O--), FFA (--Cl--), DG (--A-), MG (--A--) and PL (-¤-) by TLC. The radioactivities of these lipid bands and total lipids (-a-) were counted. The procedures of lipid extraction and separation into the lipid classes were shown in Materials and Methods. Abbreviations are shown in Table 1.
Time (h)
Fig. 4. Time course changes of radioactivity distribution in the phospholipid classes of carp plasma after force-feeding [l-‘4C]triolein. The radioactivities of PC (-_O-), SPM (--A--) and PI, PS, PE and LPC (--V--) in carp plasma were counted as described in Fig. 3. The details are shown in Materials and Methods, and abbreviations are shown in Table I.
I
*W-M
-+-
Llk -+-
HDL -I-
Others --I
72
5
(h)
--
40
50
60
70
80
Elutlon volume (ml)
Fig. 5. HPGC elution profile of protein and time course changes of radioactivity distributions in the plasma lipoprotein fractions separated by HPGC after force-feeding [I-“Cltriolein. Upper panel shows the elution profile of carp plasma protein at 3 hr after force-feeding labelled compound as an example. The time given for the lower panel represents the time after force-feeding labelled compound. Each 50 ~1 of plasma taken at selected times after dosing [I-r4C]triolein was loaded onto the combination of TSK-GEL G5OOOPWand G4OOOPW.and the effluent from post column was directly monitored at 280 nm. The radioactivities in the eluents of each 1 ml fraction were counted. Details are shown in Materials and Methods. Others
I
2 0 0
12
20
28
40
72
Time (h)
Fig. 6. Time course changes of radioactivity distributions in each lipid class of carp plasma lipoprotein fractions separated by HPGC after force-feeding (I-r4C]triolein. Carp plasma taken at various intervals were loaded onto the combination of TSK-GEL GSOOOPWand G4OOOPW,and VLDL, LDL, HDL and other fractions were obtained as shown in Fig. 5. Lipids extracted with chloroform-methanol from each lipoprotein fraction were separated by TLC into TG (-_O-), FFA (--o--), MG and DG (--A--) and PL (-¤-) fractions, and the radioactivities of their fractions and total lipids (-•--) were measured as described in Materials and Methods.
49
Absorption and transport of TG and PC high density lipoprotein (HDL) band until 9 hr, and its activity was almost constant. Radioactivities in Band III; low density lipoprotein, (LDL,) and Band II; low density lipoprotein, (LDL,) increased from 12 to 20 hr, followed by relatively constant values. The radioactivity in Band IV (VLDL) was very low during the experimental period. Figures 3 and 4 show the time course changes of radioactivity distribution in the lipid classes of carp plasma after force-feeding with [ l-14C]triolein. Total radioactivity of carp plasma lipids rapidly increased up to 28 hr,
and then showed almost constant activity. The radioactivity in TG quickly increased until 20 hr, followed by a gradual decrease. Radioactivity in PL slowly increased up to 28 hr, mainly in the form of PC and sphingomyelin. Figure 5 shows the time course changes of radioactivity distributions of carp plasma lipoprotein fractions separated by HPGC after dosing of [1-“Qriolein. The radioactivity was mainly detected in both HDL and LDL fractions at 12 and 20 hr, however, main activity was found in HDL fraction at 28, 48 and 72 hr. Figure 6 shows I-1
Time (h)
Fig. 7. Time course changes of radioactivity distributions in carp plasma lipoprotein bands by PAGE after force-feeding [l-‘4C]dioleyl phosphatidylchohne. The radioactivities of whole carp plasma (-a-) and lipoprotein Bands: Band I, HDL (-_O-); Band II, LDL, (-a-); Band III, LDL, (--A--); and Band IV, VLDL (-A--) were measured as shown in Fig. 2.
“036912
20
28 Time (h)
48
72
Fig. 8. Time course changes of radioactivity distributions in the lipid classes of carp plasma after force-feeding [I-r4C]dioleyl PC. The radioactivities of CE and WE (--a --), TG (-_O-), DG (-A-), FFA (--O--), MG (--A--), PL (-¤---) and total lipids (-a-) were counted as described in Fig. 3. Abbreviations are shown in Table I.
Tlma (h) Fig. 9. Time course changes of radioactivity distribution in the phospholipid classes of carp plasma after force-feeding Il. .i4C]dioleyl PC. The radioactivities of PL (-¤-), PC (-_O-) and PE, PI, PS, SPM and LPC (--V--) in carp plasma were counted as described in Fig. 3. Abbreviations are shown in Table I.
50
NORIAKI IIJIMAet al.
the time course changes of radioactivity distributions of various lipid classes in carp plasma lipoprotein fractions separated by HPGC. At 12 hr after dosing [l-‘4C]triolein, incorporation of radioactivity was almost the same in all VLDL, LDL, HDL and other fractions, but thereafter the radioactivity in the HDL fraction was higher than those of VLDL, LDL and other fractions until 72 hr. In the HDL fraction, the radioactivity was mainly associated with TG until 28 hr, whereas the radioactivity in PL gradually increased at the latter stage of lipid absorption (48-72 hr). The distribution of radioactivities in the lipid classes of various tissues in carp after force-feeding with [l-‘4C]triolein are shown in Table 1. A high amount of radioactivity remained in the intestinal contents (28.4%). Moreover, high radioactivities were distributed in other tissues (19.5%) and gills (17.9%), followed by intestine (9.2%), muscle (8.8%), and hepatopancreas (3.9%), mainly as TG and PL.
3 .r
> 5
Incorporation of [l-‘4C]dioleyl PC into plasma lipids and lipoproteins
Figure 7 shows the radioactivity distributions in carp plasma lipoprotein bands by PAGE after force-feeding [I-i4C]dioleoyl PC. Plasma radioactivity rapidly increased up to 20 hr, followed by a gradual decrease until 72 hr. The radioactivity in Band I (HDL) comprised over 70% of plasma radioactivity at any interval of time. The maximum incorporation of radioactivities in VLDL and LDL, bands were found at 20 and 28 hr, respectively. Time course changes of radioactivity distribution in the lipid classes of carp plasma after dosing of [l-‘4C]dioleoyl PC are shown in Figs 8 and 9. About 60% of total radioactivity was always distributed in TG fraction during the experimental period. The radioactivity in FFA fraction progressively increased with a maximum around 12 and 20 hr, and then decreased. Incorporation of radioactivity into PL fraction reached maximum at 28 hr, and its main radioactivity was detected in PC. Time course changes of positional distribution of radioactivity in carp plasma PC after force-feeding [l-‘4C]dioleoyl PC are shown in Fig. 10. The radioactivity was detected only in the 2-position of PC at 6 hr after dosing; however, the incorporation of radioactivity into the l-position of PC was approximately twice that in the 2-position
5
6
5
Ir 40
50
60
70
Fig. 11. Elution profile of carp plasma protein and time course changes of radioactivity distributions in carp plasma lipoprotein fractions separated by HPGC after force-feeding [l-‘4C]dioleyl PC. Details are shown in Fig. 5.
from the maximum lipid absorption (20 hr) to 72 hr after dosing. Figure 11 shows the time course changes of radioactivity distributions in carp plasma separated by HPGC after force-feeding [ l-‘4C]dioleyl PC. Although the main radioactivity was distributed in HDL fraction during the experimental period, low activity was also recognized in VLDL and LDL fractions. Figure 12 shows the time course changes of radioactivity distributions in the various lipid classes of each lipoprotein fraction separated by HPGC after force-feeding [ l-‘4C]dioleyl PC. At 12 hr after feeding, radioactivity was detected almost equally in all VLDL, LDL and HDL fractions. However, at the peak of lipid absorption (20 hr), the radioactivity in 0
lposition
m
2position
n
0
6
12
20
80
Elutlon volume (ml)
26
46
72
Time (h)
Fig. 10. Time course changes of radioactivity distribution in the fatty acids moieties of PC in carp plasma after force-feeding [I-‘4C]dioleyl PC. Plasma PC at the selected intervals after dosing of labelled PC were hydrolysed with phospholipase A,, and the radioactivities of I-position (I-acyl 1ysoPC) and 2-position (free fatty acids) of plasma PC were counted as described in Materials and Methods.
J+cpp-
483.59
85.934.5
100.6 229.8 567.3 63.5 1059.2 15,670.O 2840.0 934.3 64.5 113.2 14,340.o Il,8201) 269.8 41.3 22.8 28.2 37,760.o 85,333.9
Total radioactivity (dpm) (%)
$I:; (71.3)
::;;
:z; (0:3)
:::;
g-i; (0:5) (3.9) (3.0) (1.0) (18.1)
P-7)
6.2 0.1
5.8 -
-
LPC 1.1 3.1 0.8 3.3 8.3 0.8 4.9 3.1 0.9 14.9 4.1 2.1
-
SPM
44.2 30.5 37.2 28.5 43.3 40.7
1.0 19.8 45.2 54.9 49.0 13.8 49.5 51.7 -
PC 0.3 1.7 2.3 5.4 2.2 4.7 3.8 4.6 4.5 7.5 4.2
PI+PS
6.8 7.5 9.5 8.6 5.9
0.5 3.0 6.1 2.3 6.2 1.0 5.2 -
PE 2.6 15.4 0.2
MG 17.9 6.4 26.6 16.0 6.1 36.5 14.3 5.8 5.4 7.4 6.0 18.2 18.4 8.1
DG 1.3 0.2 0.2
-
ALC 23.1 0.8 0.8 0.5 6.4 0.7 5.3 12.6 13.3 0.2 2.0
FFA 51.2 61.0 5.8 6.9 21.8 10.7 1.1 14.1 32.5 37.0 29.5 4.9 16.9 24.3
TG
Distribution of radioactivity in the lipid classes (%)
0.z
-
2.4 1.7 4.9 0.9 -
CE+ WE
1.0 4.5 10.1 12.8 1.8 36.8 4.8 11.5 4.9 10.7 9.0 1.0 7.1
Others1
‘Carp used was 496.02 g of bcdy weight. tOthertissues containskeleton. head. fins, scales and drips. jOthers contain galactolipids, diacylglycerykthers. fatty aldehydes and hydrocarbons. Abbreviations: LPC, lysophosphatidykholines; SPM, sphyngomyelins; PC, phosphatidykholines; PI, phosphatidylioositols; PS, phosphatidylserines; PE, phosphatidykthanolarnines; MG, monoglycerides; DG, diglycerides; ALC, free fatty alcohols; FFA, free fatty acids; TG. triglycxxides; CE, cholesterol esters; WE, wax esters.
SlIllI
zother tissuest Whole body
Adipose tissue Gonad Gill GZtlCbMdtX Brain Musdc !&in Swim-bladder
6.07 7.22 1.86 9.80 21.55 5.17 13.60 1.34 0.83 218.17 33.26 2.59 4 (ml) 2.30 0.84 154.49 483.59
-
Lipid content (ms)
Tabk 1. Distribution of radioactivity in the lipid classes of various tissues of carp at 72 hr after force-feeding [I-“C@iokin*
Tissue tight (=t) (I!)
NORIAKIIIJIMAet al.
52
HDL was about two to three times higher than those in VLDL and LDL, and thereafter almost all of radioactivity was found in HDL fraction. It was found that the main radioactivity in HDL fraction was TG. The distribution of radioactivities in the lipid classes of various tissues in carp at 72 hr after force-feeding [l-‘4C]dioleoyl PC is shown in Table 1. As shown in the column of lipid content, about 60% of whole body lipids were distributed in other tissues, followed by muscle (16.2%) and skin (8.7%). However, high radioactivity was detected in the intestine (28.5%), gill (25.6%) and other tissues (18.8%) in the form of PC. DISCUSSION
In fish, the digestibility of dietary lipids seems to depend on the environmental water temperature (Leger, 1985; Henderson and Tocher, 1987). At high water temperature (20-25°C) in summer season (July and August), TG hydrolase activity in carp intestinal fluid reached maximum around 9 hr after dosing soybean oil, and the peak of lipid absorption was recognized around 9 and 12 hr after dosing 14C labelled fatty acids (Robinson and Mead, 1973; Kayama and Iijima, 1976; Iijima, Aida and Kayama, unpublished data) or TG (Kayama and Iijima, 1976). On the other hand, at low water temperature (12-15°C) in winter, intraluminal TG hydrolase activity in carp very slowly increased up to 24 hr after force-feeding soybean oil (Fig. l), and maximum radioactivity in blood plasma continued from 28 and 72 hr after dosing [l-14C]triolein (Fig. 2). Moreover, high radioactivity still remains unabsorbed in the intestinal contents even at 72 hr (Table 1). It was assumed from the above results that in winter (November and December), dietary TG was hydrolysed very slowly in the intestinal lumen according to the delay of lipase secretion in the intestinal
lumen. The products, MG and FFA, were mainly reconstituted into TG and PL in the intestinal mucosa (Figs 3 and 4), followed by the release as lipoproteins into the blood circulatory system (Figs 2, 5 and 6). Recently, we separated carp plasma lipoproteins into four fractions, d < 1.006 g/ml, d = 1.006-l .063 g/ml, d = 1.063-1.125 g/ml and d = 1.125-1.21 g/ml, by their density intervals, and these four lipoprotein fractions corresponded to lipoprotein bands: Band IV (d < 1.006 g/ml), Band III (d = 1.006-1.063 g/ml), Band II (d = 1.063-1.125 g/ml) and Band I (d = 1.125-1.21 g/ml), which by PAGE were assumed to be VLDL, intermediate density lipoprotein (IDL, LDL,), LDL, and HDL, respectively. Therefore, as shown in Fig. 2, the lipoprotein bands, Band IV, Band III, Band II and Band I, were tentatively named as VLDL, LDL,, LDL, and HDL, respectively. However, we must further investigate the relationship of carp plasma lipoprotein fractions separated by sequential ultracentrifugation, PAGE and HPGC, as described previously (Hermier et al., 1985; Babin, 1987). When [l-‘4C]triolein was fed to carp, only a little radioactivity was detected in lipoprotein Band IV (VLDL) by PAGE (Fig. 2), and most of the radioactivity was detected in Band III (LDL,), Band II (LDL,) and Band I (HDL). Moreover, from 12 to 72 hr after feeding, high radioactivity was detected in both LDL and HDL fractions (Figs 5 and 6). It was considered from the above results that under the very slow absorption in winter, dietary TG were mainly released into the blood circulatory system in the form of LDL,-, LDL,- and HDL-like particles smaller than VLDL. In mammals, it was recognized that absorbed polyene-PC was incorporated preferentially into the HDL fraction of plasma (Zierenberg et al., 1979; Zierenberg and Grundy, 1982). Although high incorporation of PC into HDL was assumed to be due to Others
::
0
12
20
28
48 Time
72
(h)
Fig. 12. Time course changes of radioactivity distributions in the lipid classes of carp plasma lipoprotein fractions separated by HPGC after force-feeding [I-14C]dioleyl PC. The radioactivities of TG (-_O-), FFA (--O--), MG and DG (--A--) and PL (-¤--) were counted as described in Fig. 6.
767,300
11,903.8
5.16 4.27 1.10 6.60 0.89 5.83 9.31 0.95 0.71 150.19 18.12 2.09 4(mI) 1.2 0.73 123.86 335.01
335.01
Sum
(%)
K; (t8:8) (95.3)
r$ (1.2)
g;
(4.7) (28.5) (1.8) (0.5) (5.7) (0.7) (I.11 (25.6) (Tr.)
0.5 0.1 3.5 0.9 3.0 0.1
LPC 0.5 9.2 0.9 0.3 0.6 5.0 0.5 1.1 2.2 20.6 5.0
1.5
SPM
*Carp used was 348.27 g of body weight. tOther tissues contain skeleton, head, tins, scales and drips. fOthers contain galactolipids, diacylglyceryl ethers, fatty aldehydes and hydrocarbons. Abbreviations are as described in Table I.
36,000 219,000 14,Ofxl 3600 436,000 5200 8200 I%,600 309 100 wIO0 21,800 2200 9000 Uoo 8400 I44,Ooo 13 1,300
Total radioactivity (dpm)
34.7 145.9 153.2 25.6 248.8 109.1 444.3 303.7 5.1 101.3 1930.0 1030.0 92.1 29.3 7.2 13.5 7230.0 I11,869.l
Lipid content (msf
Intestinal content Intestine Kidney Spleen Hepstopancreas Adipose tissue Gonad Gil1 Gall-bladder Brain Muscle Skin Swim-btadder Ptasma Erythrocyte Heart other tissuest whole bcdy
-
Tissue we&h1 (wet) tSf
61.2 58.1 45.8 34.6 64.2 55.9 49.3 42.4 33.5 8.1 47.2 59.6
57.8
PC 63.4 70.2 2.6 3.6 4.8 5.0 4.0 5.0 3.4 5.6 2.3 3.2 3.6
PI+PS 2.2 11.5 4.8 4.7 9.1 2.0 4.6 7.1 1.4 6.6 0.4 6.1 3.0 6.8
PE
DG
2.7 0.5 1.7 8.9 15.5 2.8 0.1 1.4 5.5 4.7 0.3 0.z 2.6 8.8 8.3 6.5 2.2 11.2 3.0 0.4 3.0
-
MG
0.5 3.1 3.5 0.1
-
ALC
3.1 4.2
2.4 1.6 0.6 0.9 2.9 6.2 4.1 10.3 5.5 3.5 10.5
FFA
8.5
9.6 4.3 17.3 34.2 51.2 5.2 30.2 11.8 29.7 38.2 3.3 16.6 12.5
17.7
TG
Distribution of radioactivity in the lipid classes (%)
0.3
0.1 I.1 -
;:: -
G -
CE+ WE
Table 2. Distribution of radioactivity in the lipid classes of various tissues of carp at 72 hr after force-feeding [I-“CJdioleyl phosphatidyIcholine*
3; 4.4
13.2 0.1 6.6 2.0 6.1 5.4 7.9 56.3
8.0 5.8
7.0 2.8 2.6
Others$
54
NORIAKIIUIMAet al.
the selective transfer of CHY-PC (Eisenberg, 1984), direct secretion of nascent HDL by the intestine may also have contributed (Glickman and Magun, 1986). The metabolic fate of dietary PC has been investigated in carp by dosing [l-i4C]dioleyl PC. In winter the absorption of dietary TG proceeded slowly in carp at 72 hr after feeding (Figs 1 and 2); however, dietary PC was more readily absorbed than TG (Fig. 7). Moreover, from the present results, three points can be mentioned about the intestinal digestion and absorption in carp: (a) the radioactivity in the fatty acids of the l-position of plasma PC was approximately two to three times higher than that in a 2-position at the peak of lipid absorption (20 hr) (Fig. 10) (in other words, dietary PC preferentially hydrolysed its 2-acyl ester bond in carp intestinal lumen); (b) dietary PC appeared mainly as TG in blood plasma (Fig. 8); and (c) a high amount of radioactivity (28.5%) was distributed in the intestinal tissues (Table 2). We assumed from the above results that dietary PC, hydrolysed into I-acyl 1ysoPC and FFA by intraluminal phospholipase A,, were absorbed in the intestinal mucosa, and mainly resynthesized into TG and PL, especially PC. Then almost all of TG was released in blood plasma mainly as HDL followed in the decreasing order of LDL and
VLDL (Figs 7, 8 and 11). On the other hand, PC might be metabolized by two pathways that were directly secreted into blood plasma as the main form of HDL (Figs 7, 11 and 12) or incorporated as the constituent of internal membrane in the enterocytes (Table 2). Moreover, we also assumed that a part of I-acyl 1ysoPC was hydrolysed to FFA and glycerylphosphorylcholine, because a major part of dietary PC-fatty acids were incorporated into plasma TG (Fig. 8). Therefore, we now propose from the present results and the previous reports (Kayama and Iijima, 1976; Iijima et al., 1985; Mankura et al., 1987; Iijima, Aida and Kayama, unpublished data) that, in carp, a part of dietary lipids (TG and PC) were secreted both as LDL and VLDL by the intestinal mucosa, but the main transport form of lipids was HDL. In carp, HDL is the predominant class of lipoproteins in the plasma (Iijima et al., 1985; Iijima, Aihara, Kayama, Okazaki and Hara, unpublished data) like other bony fishes (Babin, 1989; Chapman,l980). This feature provides HDL with an important role in the transport of lipids. Leger (1985) proposed that high HDL levels may be due either to a low degradation rate or to a high hepatic synthesis of nascent particles or to a fast degradation of the very low density particles by lipoprotein lipase leading to release of apoprotein constituting the HDL. Moreover, the fact that CHY and VLDL are always very low in the plasma could be in favour of the last two hypotheses. Black and Skinner (1986) investigated the relationship between the concentrations of plasma lipoproteins and the activities of lipoprotein lipase and salt-resistant lipase in rainbow trout, and they suggested that HDL served a role similar to that of VLDL in the transport and uptake of fatty acids for energy production besides its function in the transport of cholesterol and as a reservoir for apoproteins. We now propose that the one reason for high HDL levels is the direct secretion of HDL from the intesti-
nal mucosa in addition to the above aspects. That is, in carp HDL seems to play a central role for the transport of dietary lipids, cholesterol, TG and PL from the enterocytes to various tissues instead of CHY and VLDL in mammals (Green and Glickman, 1981). It was recognized that also in the fishes (Babin, 1989; Black and Skinner, 1986), both lipoprotein lipase and salt-resistant lipase, which correspond to mammalian hepatic triglyceride lipase (Smith and Powell, 1984; Kinunnen, 1984), were present in numerous tissues: red and white muscle, heart, brain, liver, adipose tissue and ovaries, and intestinal lipoproteins were hydrolysed by these lipases. Released free fatty acids were incorporated into various tissues in the same manner as in mammals. It was assumed that also in carp, dietary lipids secreted as HDL, LDL and VLDL in blood plasma were hydrolysed by lipoprotein lipase or hepatic TG lipase (salt-resistant lipase), and then released fatty acids were taken up by various tissues, especially gill, muscle and other tissues (head, bone, fins etc.) mainly as PC and TG (Tables 1 and 2); however, dietary TG and PC were finally deposited as PC rather than TG in the whole body of carp in winter. REFERENCES
Arnesjo B., Nillson A., Barrowman J. and Borgstrom B. (1969) Intestinal digestion and absorption of cholesterol and lecithin in the human. &and. J. Gastroenterol. 4, 653-665.
Babin P. J. (1987) Plasma lipoprotein and apoprotein distribution as a function of density in the rainbow trout (Sabno gairdneri). Biochem. J. 246, 425-429.
Babin P. J. (1989) Plasma lipoproteins in fish. J. Lipid Res. 30, 467-489.
Baxter J. H. (1966) Origin and characteristics of endogeneous lipid in thoracic duct lymph in rat. J. Lipid Res. 7, 158-166.
Black D. and Skinner E. R. (1986) Features of the lipid transport system of fish as demonstrated by studies on starvation in the rainbow trout. J. camp. Physiol. 156B, 497-502.
Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Physiol. 37, 91 l-917. Boucrot P. (1972) Is there an entero-hepatic circulation of the bile phospholipids? Lipids 7, 282-288. Carey M. C., Small D. M. and Bliss C. M. (1983) Lipid digestion and absorption. Ann. Rev. Physiol. 45.651-677. Chapman M. J. (1980) Animal lipoproteins: chemistry, structure, and comparative aspects. J. Lipid Res. 21, 789-853.
Eisenberg S. (1984) High density lipoproteins. J. Lipid Res. 25. 1017-1059.
Folch J., Lee M. and Sloane Stanly G. N. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Glickman R. M. and Magun A. M. (1986) High-density lipoprotein formation by the intestine. Methodr Enzymol. 129, 519-536.
Green P. H. R. and Glickman R. M. (1981) Intestinal lipoprotein metabolism. J. Lipid Res. 22, 1153-l 173. Hara I. and Okazaki M. (1986) High performance liquid chromatography of serum lipoproteins. Methoak Enzymol. 129, 57-78. Henderson R. J. and Tocher D. R. (1987) The lipid composition and biochemistry of freshwater fish. Progr. Lipid Res. 26, 281-347.
Absorption and transport of TG and PC Hermier D., Forgez P. and Chapman M. J. (1985) A density of the lipoprotein and apoprotein distribution in the chicken, Gallus domesticus. Biochim. biophys. Acta 836, 105-I 18.
Iijima N. and Zama K. (1979) Influence of oxidized lipids on the lipid metabolism in the fish. II. Absorption and metabolism of palmitic acid-l-14C in carp fed with oxidized lipids. Bull. Fat. Fish. Hokkaido Univ. 30, 84-91. Iijima N., Zama K. and Kayama M. (1983) Effect of the oxidized lipids on the metabolic pathway of lipid biosynthesis in the intestine of carp. Bull. Jpn Sot. Sci. Fish. 49, 1465-1470.
Iijima N., Kayama M., Okazaki M. and Hara I. (1985) Time course changes of lipid distribution in carp plasma lipoprotein after force-feeding with soybean oil. Bull. Jpn Sot. Sci. Fish. 51, 467-471. Kayama M. and Tsuchiya Y. (1959) Fat metabolism in the fish. II. Intestinal absorption and distribution study of oil in the carp, (Cyprinus carpio Linne). Tohoku J. Agric. Res. 10, 229-236.
Kayama M. and Tsuchiya Y. (1965) Incorporation of C4-labelled acetate into lipid classes of liver oil by intact leopard shark. Tohoku J. Agric. Res. 15, 259-267. Kayama M. and Iijima N. (1976) Studies of lipid transport mechanism in the fish. Bull. Jpn Sot. Sci. Fish. 42, 987-996.
Kinunnen
P. K. J. (1984) Hepatic endothelial lipase. In
Lipases (Edited by Borgstrom B. and Brockman H. L.),
307-328. Elsevier Science Publishers, Amsterdam. Leger C. (1985) Digestion, absorption and transport of lipids. In Nutrition and Feeding in Fish (Edited by Cowey C. B., Mackie A. M. and Bell J. G.), pp. __ 299-331. Academic Press, London. Mankura M.. Iiiima N.. Kavama M. and Aida S. (1987) Plasma transport form and metabolism of dietary fatty alcohol and wax ester in carp. Nippon Suisan Gakkaishi 53, 1221-1230.
Mansbach
C. M. II. (1977) The origin of chylomicron
55
phosphatidylcholine in the rat. J. clin. Invest. 90, 41 l-420. Gckner R. K., Hughes F. B. and Isselbacher K. J. (1969) Very low density lipoproteins in intestinal lymph: origin, composition, and role in lipid transport in the fasting state. J. clin. Invest. 48, 2079-2088. Patton J. S., Nevenzel J. C. and Benson A. A. (1975) Specificity of digestive lipases in hydrolysis of wax esters and triglycerides studied in anchovy and other selected fish. Lipia!s 10, 575-583. Patton J. S., Haswell M. S. and Moon T. W. (1978) Aspects of lipid synthesis, hydrolysis, and transport studied in selected Amazon fish. Can. J. Zool. 56, 787-792. Robinson J. S. and Mead J. F. (1973) Lipid absorption and deposition in rainbow trout (Salmo gairdneri). Can. J. Biochem. 51, 1050-1058. Scow R. O., Stein Y. and Stein 0. (1967) Incorporation of dietary lecithin and lysolecithin into lymph chylomicrons in the rat. J. biol. Chem. 242. 4919-4924. Sire M.-F. Lutton C. and Vernier J.-M. (1981) New views on intestinal absorption of lipids in teleostean fishes: an ultrastructural and biochemical study in the rainbow trout. J. Lipid Res. 22, 81-94. Smith L. C. and Powell H. J. (1984) Lipoprotein lipase. In Lipases (Edited by Borgstrom B. and Brockman H. L.), pp. 263-305. Elsevier Science Publishers, Amsterdam. Tocher D. R. and Sargent J. R. (1984) Studies on triacylglycerol, wax ester and sterol ester hydrolase in the intestinal caeca of rainbow trout (Salmo gairdneri) fed diet rich in triacylglycerols and wax esters. Comp. Biochem. Physiol. 77B, 561-571.
Zierenberg 0. and Grundy S. M. (1982) Intestinal absorption of polyenephosphatidylcholine in man. J. Lipid Res. 23, 1136-l 142. Zierenberg O., Odenthal J. and Betzing H. (1979) Incorporation of polyenephosphatidylcholine into serum lipoproteins after oral or intravenous administration, Atherosclerosis 34, 259-276.
0300-9629/90 $3.00 + 0.00 0 1990 PergamonPressplc
1990
INTESTINAL ABSORPTION AND PLASMA TRANSPORT OF DIETARY TRIGLYCERIDE AND PHOSPHATIDYLCHOLINE IN THE CARP (CYPRINUS CARPI0) NORIAKI IIJIMA,* SATOSHI AIDA,~
MITSUMASA MANKURA~
MITSU KAYAMA*
*Faculty of Applied Biological
Science, Hiroshima University, Shitami, Saijo-cho, Higashi-Hiroshima 724, Japan. Telephone: 0824-22-7 I 11; YHiroshima Prefectural Fisheries Experimental Station, Ondo-cho, Akigun, 737-12, Japan. Telephone: 0823-51-2171; and $Ikeda Tohka Industries Co. Ltd, Fukuyama 720, Japan. Telephone: 0849-54-5733 (Received
10 October 1989)
Abstract-l.
Intraluminal TG hydrolase activity of carp very slowly increased until 24 hr after dosing soybean oil. 2. In oiuo metabolic fates of dietary TG and phosphatidylcholine (PC) were investigated in carp, by measuring radioactivities of plasma lipids and lipoproteins after force-feeding [I-‘%]triolein or [l-‘4C]dioleyl PC. 3. After dosing [I-%]triolein, radioactivity was mainly distributed in carp plasma HDL and LDL fractions as TG. 4. When [l-‘4C]dioleyl PC was fed, a part of radioactivity was found both in carp plasma VLDL and LDL fractions; however, the major radioactivity distributed in HDL fraction as TG. At 20 hr after dosing, radioactivity in l-position of plasma PC was more than twice that in 2-position.
INTRODUffION
cation in blood plasma (Robinson and Mead, 1973; Kayama and Iijima, 1976; Patton et al., 1978); however, a major part of FFA are reconstituted into TG and then released as CHY and VLDL-like particles (Sire et al., 1981). Recently, we postulated that in carp, TG hydrolytic products were secreted as HDL-; LDL- and VLDL-like particles (mainly HDL) into the blood circular system after resynthesizing TG and PL in the intestinal mucosa (Iijima and Zama, 1979; Iijima et al., 1983; Iijima et al., 1985; Mankura et al., 1987; Iijima, Aida and Kayama, unpublished data). Several investigations have been made on the absorption of TG in fish; however, there is now some controversy over the above findings. Moreover, the mechanisms of digestion, absorption and transport of dietary PC have not yet been studied in fish. The present study was performed to examine the metabolic fate of dietary TG and PC in carp by force-feeding [ l-‘4C]dioleyl PC, collecting blood via the tube cannulated into dorsal aorta, and assaying the radioactivities of plasma lipids and lipoproteins.
In mammals, the mechanisms of digestion and absorption of TG have been studied in great detail. Dietary TG, partially hydrolysed to FFA and MG by pancreatic lipase in the intestinal lumen, are absorbed in the intestinal mucosa. In the intestinal mucosa, FFAs and MGs are mainly reconstituted into TG and PL which are associated with apoproteins, and released as chylomocron (CHY) and (VLDL) in the lymphatic system (Green and Glickman, 1981). On the other hand, dietary PL, especially PC, are hydrolysed to FFA and I-acyl 1ysoPC by pancreatic phospholipase A, activated by protease in the intestinal lumen (Carey et al., 1983; Arnesjo et al., 1969). FFA absorbed in the intestinal mucosa are metabolized as described above. However, several routes have been proposed about the metabolism of 1ysoPC in the intestinal mucosa: (a) re-esterification with FFA to form PC which can be utilized by CHY formation or for the constituent of intracellular membranes (Scow ef al., 1967; Mansbach, 1977); (b) complete hydrolysis and use of the released FFA of 1ysoPC for TG synthesis (Baxter, 1966; Ockner et al., 1969); and (c) direct absorption of 1ysoPC into the portal circulation (Boucrot, 1972). In fishes, several differences have been proposed for the digestion, absorption and transport of dietary TG compared with that in mammals: (a) some marine species completely hydrolysed dietary TG to FFA and glycerols (Patton et al., 1975; Leger, 1985), but in rainbow trout, it was partially hydrolysed to FFA, MG and DG in the intestinal lumen (Tocher and Sargent, 1984); and (b) part of FFA absorbed in the intestinal mucosa is directly released without esterifi-
MATERIALS AND METHODS
Isotope
L-a-[l-‘4C]dioleoyl phosphatidylcholine([l-‘4C]dioleyl PC, 114.0 mCi/mmol) and Il-14Cltriolein (103.5 mCiimmo1) were purchased from New &gland Nuclear througd the Nippon Isotope Association. NCS-solubilizer was purchased from Amersham Inc. All other chemicals were of the guaranteed grade available from commercial sources.
Carp (Cyprinus carpio), of 500-600 g body weight, were maintained in a large outdoor tank with pellet No. 5P for 45
NORIAKIIIJIMA
46
carp (Nippon Haigoshiryo December.
Co. Ltd) from November to
Intraluminal TG hydrolysis
Carp were starved for 2 days and then administered soybean oil (0.5 ml/100 g body weight) under anesthesia. After swimming freely in a tank, carp were killed at 0, 3, 6, 9, 12 and 24 hr, and the intestinal tracts were removed. The intestinal contents were squeezed and washed with physiological saline solution in an ice-cold centrifuge tube, and centrifuged at 27,000g for 20min. After removal of the upper lipid layer, the infranatant was used as the crude enzyme extract. 0.5ml of the crude enzyme extract at various intervals was incubated at 0.25 PCi of [I-i4C]triolein dissolved in 1 mg non-labelled triolein. The enzyme reaction was stopped by the addition of three drops of 3 N HCl, and then the lipids were extracted with diethyl ether according to the modified method of Patton et al. (1975). The residual lipids, after being removed from the solvent under reduced pressure. were separated by a TLC plate coated with silica
100 I-
et al.
gel 6OG (Merck), which was first developed with diethyl ether up to 2cm from origin, followed by the solvent system of benzene-chloroform-formic acid (70: 30: 2, v/v/v). The lipid bands were visualized with 0.01% (w/v) I-hydroxy-l,3,6-pyrenetrisulfonic acid t&odium salt (Kodak)-methanol under ultraviolet light, and identified with authentic lipid standards (monoolein, diolein, triolein and oleic acid). The separated lipid bands were scraped directly into the scintillation vials, and suspended in IO ml of toluene-based scintillation cocktail (0.2 g of 1,4&s[2-(5phenyloxazolyl)]-benzene and 4 g of 2,5-diphenyloxazole per 1000 ml of toluene) and 0.2 g of Cab-0-Sil (thixotropic gel powder, Packard). The radioactivity was measured with a liquid scintillation spectrometer (LSC-903, Aloka Co. Ltd). In vivo experiment The operation of dorsal aorta cannulation and force-feeding with labelled compounds were described elsewhere (Kayama and Iijima, 1976; Kayama and Tsuchiya, 1959). Each cannulated carp was force-fed 20 PCi of [1-‘4C]dioleoyl PC or 50 FCi of [l-‘4C]triolein, dissolved in 0.5 ml of carp body lipids, after anesthetization with 0.01% MS222 (ethyl m-aminobenzoate methane sulfonate, Nakarai Tesque). Then the fish was transferred into each aquarium in a constant water temperature (20°C). Blood (0.5 ml) was collected from the cannulated tube at intervals of 0, 3, 6, 9, 12, 28, 48 and 72 hr after dosing of labelled compounds. Plasma was obtained by centrifugation at l5OOg for IOmin andNaN, was added at a final concentration of 0.01%. Analysis of plasma lipids and lipoproteins
Tim
(h)
Fig. 1. In vitro TG digestion in the intestinal lumen of carp in winter. The intestinal fluid obtained at various intervals after force-feeding soybean oil was incubated for 30 min at 20°C with [l-‘4C]triolein. Then the radioactivities of MG (--n--), DG (-A-), FFA (--0 --) and TG (-a-) were determined and expressed as mole percentage as described in Materials and Methods.
Each 25 ~1 of plasma at various intervals was mixed with 150~1 of NCA-solubilizer in a scintillation vial, and then IO ml of scintillation cocktail (ACS-II, Amersham) was added after standing for 30min at room temperature. Plasma lipids were extracted with chloroform-methanol by the modified method of Folch et al. (1957). Plasma lipid classes were separated by TLC. The TLC plate was developed with the two step solvent systems of diethyl ether and n -hexane-diethyl ether-acetic acid (85 : I5 : 1, v/v/v), respectively, as described above. Phospholipids were also separated with TLC coated with kiesel gel HF containing 0.01% Na,CO, . The plate was developed with chloroformmethanol-acetic acid-H,0 (25: I5 : 4: 2, v/v/v/v). The separated lipid bands were scraped directly into the scintillation t-1 (VLDL) III(LDLl1
IV
II(LDL2) I(HDL)
Tlmfz(h)
Fig. 2. Time course changes of radioactivity distribution in carp plasma lipoprotein bands by PAGE after force-feeding [I-‘4C]triolein. Whole carp plasma at various intervals was separated by PAGE into four lipoprotein bands: Band I, HDL (-_O-); Band II, LDL, (--0-k Band III, LDL, (--A--); and Band IV, VLDL (-A-], The radioactivities of their bands and whole plasma (-•--) were counted as described in Materials and Methods.
Absorption and transport of TG and PC vials, and the radioactivity was measured with a liquid scintillation spectrometer. The scintillation cocktail for non-polar and polar lipids was toluene-based scintillator, containing Cab-O-%1 and ACS-II, respectively. The radioactivities of carp plasma lipoproteins at various intervals were analysed by polyacrylamide gel electrophoresis (PAGE) (Kayama and Iijima, 1976; Mankura et al., 1987) or high performance gel-filtration chromatography (HPGC) (Hara and Okazaki, 1986). Each plasma (50 ~1) was separated into four lipoprotein bands: Band I, Band II, Band III and Band IV, by PAGE. Moreover, each plasma (40 ~1) was loaded onto the high performance liquid chromatograph (HLC 803D, Toso Co. Ltd.), equipped with the combination of TSK gel G4OOOPW(7.5 x 600 mm, Toso Co. Ltd) and TSK gel G5OOOPW(7.5 x 600 mm, Toso Co. Ltd) protected with a guard column (TSK gel PWH), and eluted with 0.15 M NaCl at a flow rate of 0.5 ml/min, by monitoring at 280nm with a spectrophotometer (UV-8 Model II, Toso Co. Ltd). The eluent directly collected into scintillation vials at 2 min intervals was suspended in 10 ml of ACS-II, and the radioactivity was measured. Analysisof tissue lipids The carp were killed after the final blood sampling at 72 hr, and then the intestinal content and various tissues were excised. Plasma lipids were extracted with chloroform-methanol. Remaining blood corpuscles were washed three times with ice-cold physiological saline solution and lyophilized. Then, the lipids of lyophilized blood corpuscles, intestinal contents and various tissues were extracted with
47
chloroform-methanol according to the method of Bligh and Dyer (1959). Total lipids were fractionated into non-polar and polar lipids by silicic acid column chromatography (Kayama and Tsuchiya, 1965), and non-polar and polar lipids were separated by TLC with a solvent system of petroleum ether-diethyl ether-acetic acid (85 : 15: 1, v/v/v) and chloroforn-methanol-acetic acid-H,0 (25 : 15 : 4 : 2, v/v/v/v), respectively. Then, the radioactivities of various lipids bands were measured, as described above. RESULTS
Intraluminal TG hydrolysis Figure 1 shows the time course changes of TG hydrolase activity by carp intestinal fluid after forcefeeding with soybean oil. TG hydrolase activity was very low until 9 hr after force-feeding with soybean oil, and then gradually increased up to 24 hr. Even at 24 hr after feeding, only 25% of TG was hydrolysed, and its hydrolytic products were MG, DG and FFA. Incorporation of [l-‘4C]triolein into plasma lipidr and lipoproteins Time course changes of radioactivity distribution in carp plasma lipoprotein bands by PAGE after
force-feeding [l-‘4C]triolein are shown in Fig. 2. Almost all of the radioactivity was found in Band I;
Time (h) Fig. 3. Time course changes of radioactivity distribution in the lipid classes of carp plasma after force-feeding [l-‘4C]triolein. Carp plasma lipids at various intervals were separated into CE and WE (--a--), TG (-_O--), FFA (--Cl--), DG (--A-), MG (--A--) and PL (-¤-) by TLC. The radioactivities of these lipid bands and total lipids (-a-) were counted. The procedures of lipid extraction and separation into the lipid classes were shown in Materials and Methods. Abbreviations are shown in Table 1.
Time (h)
Fig. 4. Time course changes of radioactivity distribution in the phospholipid classes of carp plasma after force-feeding [l-‘4C]triolein. The radioactivities of PC (-_O-), SPM (--A--) and PI, PS, PE and LPC (--V--) in carp plasma were counted as described in Fig. 3. The details are shown in Materials and Methods, and abbreviations are shown in Table I.
I
*W-M
-+-
Llk -+-
HDL -I-
Others --I
72
5
(h)
--
40
50
60
70
80
Elutlon volume (ml)
Fig. 5. HPGC elution profile of protein and time course changes of radioactivity distributions in the plasma lipoprotein fractions separated by HPGC after force-feeding [I-“Cltriolein. Upper panel shows the elution profile of carp plasma protein at 3 hr after force-feeding labelled compound as an example. The time given for the lower panel represents the time after force-feeding labelled compound. Each 50 ~1 of plasma taken at selected times after dosing [I-r4C]triolein was loaded onto the combination of TSK-GEL G5OOOPWand G4OOOPW.and the effluent from post column was directly monitored at 280 nm. The radioactivities in the eluents of each 1 ml fraction were counted. Details are shown in Materials and Methods. Others
I
2 0 0
12
20
28
40
72
Time (h)
Fig. 6. Time course changes of radioactivity distributions in each lipid class of carp plasma lipoprotein fractions separated by HPGC after force-feeding (I-r4C]triolein. Carp plasma taken at various intervals were loaded onto the combination of TSK-GEL GSOOOPWand G4OOOPW,and VLDL, LDL, HDL and other fractions were obtained as shown in Fig. 5. Lipids extracted with chloroform-methanol from each lipoprotein fraction were separated by TLC into TG (-_O-), FFA (--o--), MG and DG (--A--) and PL (-¤-) fractions, and the radioactivities of their fractions and total lipids (-•--) were measured as described in Materials and Methods.
49
Absorption and transport of TG and PC high density lipoprotein (HDL) band until 9 hr, and its activity was almost constant. Radioactivities in Band III; low density lipoprotein, (LDL,) and Band II; low density lipoprotein, (LDL,) increased from 12 to 20 hr, followed by relatively constant values. The radioactivity in Band IV (VLDL) was very low during the experimental period. Figures 3 and 4 show the time course changes of radioactivity distribution in the lipid classes of carp plasma after force-feeding with [ l-14C]triolein. Total radioactivity of carp plasma lipids rapidly increased up to 28 hr,
and then showed almost constant activity. The radioactivity in TG quickly increased until 20 hr, followed by a gradual decrease. Radioactivity in PL slowly increased up to 28 hr, mainly in the form of PC and sphingomyelin. Figure 5 shows the time course changes of radioactivity distributions of carp plasma lipoprotein fractions separated by HPGC after dosing of [1-“Qriolein. The radioactivity was mainly detected in both HDL and LDL fractions at 12 and 20 hr, however, main activity was found in HDL fraction at 28, 48 and 72 hr. Figure 6 shows I-1
Time (h)
Fig. 7. Time course changes of radioactivity distributions in carp plasma lipoprotein bands by PAGE after force-feeding [l-‘4C]dioleyl phosphatidylchohne. The radioactivities of whole carp plasma (-a-) and lipoprotein Bands: Band I, HDL (-_O-); Band II, LDL, (-a-); Band III, LDL, (--A--); and Band IV, VLDL (-A--) were measured as shown in Fig. 2.
“036912
20
28 Time (h)
48
72
Fig. 8. Time course changes of radioactivity distributions in the lipid classes of carp plasma after force-feeding [I-r4C]dioleyl PC. The radioactivities of CE and WE (--a --), TG (-_O-), DG (-A-), FFA (--O--), MG (--A--), PL (-¤---) and total lipids (-a-) were counted as described in Fig. 3. Abbreviations are shown in Table I.
Tlma (h) Fig. 9. Time course changes of radioactivity distribution in the phospholipid classes of carp plasma after force-feeding Il. .i4C]dioleyl PC. The radioactivities of PL (-¤-), PC (-_O-) and PE, PI, PS, SPM and LPC (--V--) in carp plasma were counted as described in Fig. 3. Abbreviations are shown in Table I.
50
NORIAKI IIJIMAet al.
the time course changes of radioactivity distributions of various lipid classes in carp plasma lipoprotein fractions separated by HPGC. At 12 hr after dosing [l-‘4C]triolein, incorporation of radioactivity was almost the same in all VLDL, LDL, HDL and other fractions, but thereafter the radioactivity in the HDL fraction was higher than those of VLDL, LDL and other fractions until 72 hr. In the HDL fraction, the radioactivity was mainly associated with TG until 28 hr, whereas the radioactivity in PL gradually increased at the latter stage of lipid absorption (48-72 hr). The distribution of radioactivities in the lipid classes of various tissues in carp after force-feeding with [l-‘4C]triolein are shown in Table 1. A high amount of radioactivity remained in the intestinal contents (28.4%). Moreover, high radioactivities were distributed in other tissues (19.5%) and gills (17.9%), followed by intestine (9.2%), muscle (8.8%), and hepatopancreas (3.9%), mainly as TG and PL.
3 .r
> 5
Incorporation of [l-‘4C]dioleyl PC into plasma lipids and lipoproteins
Figure 7 shows the radioactivity distributions in carp plasma lipoprotein bands by PAGE after force-feeding [I-i4C]dioleoyl PC. Plasma radioactivity rapidly increased up to 20 hr, followed by a gradual decrease until 72 hr. The radioactivity in Band I (HDL) comprised over 70% of plasma radioactivity at any interval of time. The maximum incorporation of radioactivities in VLDL and LDL, bands were found at 20 and 28 hr, respectively. Time course changes of radioactivity distribution in the lipid classes of carp plasma after dosing of [l-‘4C]dioleoyl PC are shown in Figs 8 and 9. About 60% of total radioactivity was always distributed in TG fraction during the experimental period. The radioactivity in FFA fraction progressively increased with a maximum around 12 and 20 hr, and then decreased. Incorporation of radioactivity into PL fraction reached maximum at 28 hr, and its main radioactivity was detected in PC. Time course changes of positional distribution of radioactivity in carp plasma PC after force-feeding [l-‘4C]dioleoyl PC are shown in Fig. 10. The radioactivity was detected only in the 2-position of PC at 6 hr after dosing; however, the incorporation of radioactivity into the l-position of PC was approximately twice that in the 2-position
5
6
5
Ir 40
50
60
70
Fig. 11. Elution profile of carp plasma protein and time course changes of radioactivity distributions in carp plasma lipoprotein fractions separated by HPGC after force-feeding [l-‘4C]dioleyl PC. Details are shown in Fig. 5.
from the maximum lipid absorption (20 hr) to 72 hr after dosing. Figure 11 shows the time course changes of radioactivity distributions in carp plasma separated by HPGC after force-feeding [ l-‘4C]dioleyl PC. Although the main radioactivity was distributed in HDL fraction during the experimental period, low activity was also recognized in VLDL and LDL fractions. Figure 12 shows the time course changes of radioactivity distributions in the various lipid classes of each lipoprotein fraction separated by HPGC after force-feeding [ l-‘4C]dioleyl PC. At 12 hr after feeding, radioactivity was detected almost equally in all VLDL, LDL and HDL fractions. However, at the peak of lipid absorption (20 hr), the radioactivity in 0
lposition
m
2position
n
0
6
12
20
80
Elutlon volume (ml)
26
46
72
Time (h)
Fig. 10. Time course changes of radioactivity distribution in the fatty acids moieties of PC in carp plasma after force-feeding [I-‘4C]dioleyl PC. Plasma PC at the selected intervals after dosing of labelled PC were hydrolysed with phospholipase A,, and the radioactivities of I-position (I-acyl 1ysoPC) and 2-position (free fatty acids) of plasma PC were counted as described in Materials and Methods.
J+cpp-
483.59
85.934.5
100.6 229.8 567.3 63.5 1059.2 15,670.O 2840.0 934.3 64.5 113.2 14,340.o Il,8201) 269.8 41.3 22.8 28.2 37,760.o 85,333.9
Total radioactivity (dpm) (%)
$I:; (71.3)
::;;
:z; (0:3)
:::;
g-i; (0:5) (3.9) (3.0) (1.0) (18.1)
P-7)
6.2 0.1
5.8 -
-
LPC 1.1 3.1 0.8 3.3 8.3 0.8 4.9 3.1 0.9 14.9 4.1 2.1
-
SPM
44.2 30.5 37.2 28.5 43.3 40.7
1.0 19.8 45.2 54.9 49.0 13.8 49.5 51.7 -
PC 0.3 1.7 2.3 5.4 2.2 4.7 3.8 4.6 4.5 7.5 4.2
PI+PS
6.8 7.5 9.5 8.6 5.9
0.5 3.0 6.1 2.3 6.2 1.0 5.2 -
PE 2.6 15.4 0.2
MG 17.9 6.4 26.6 16.0 6.1 36.5 14.3 5.8 5.4 7.4 6.0 18.2 18.4 8.1
DG 1.3 0.2 0.2
-
ALC 23.1 0.8 0.8 0.5 6.4 0.7 5.3 12.6 13.3 0.2 2.0
FFA 51.2 61.0 5.8 6.9 21.8 10.7 1.1 14.1 32.5 37.0 29.5 4.9 16.9 24.3
TG
Distribution of radioactivity in the lipid classes (%)
0.z
-
2.4 1.7 4.9 0.9 -
CE+ WE
1.0 4.5 10.1 12.8 1.8 36.8 4.8 11.5 4.9 10.7 9.0 1.0 7.1
Others1
‘Carp used was 496.02 g of bcdy weight. tOthertissues containskeleton. head. fins, scales and drips. jOthers contain galactolipids, diacylglycerykthers. fatty aldehydes and hydrocarbons. Abbreviations: LPC, lysophosphatidykholines; SPM, sphyngomyelins; PC, phosphatidykholines; PI, phosphatidylioositols; PS, phosphatidylserines; PE, phosphatidykthanolarnines; MG, monoglycerides; DG, diglycerides; ALC, free fatty alcohols; FFA, free fatty acids; TG. triglycxxides; CE, cholesterol esters; WE, wax esters.
SlIllI
zother tissuest Whole body
Adipose tissue Gonad Gill GZtlCbMdtX Brain Musdc !&in Swim-bladder
6.07 7.22 1.86 9.80 21.55 5.17 13.60 1.34 0.83 218.17 33.26 2.59 4 (ml) 2.30 0.84 154.49 483.59
-
Lipid content (ms)
Tabk 1. Distribution of radioactivity in the lipid classes of various tissues of carp at 72 hr after force-feeding [I-“C@iokin*
Tissue tight (=t) (I!)
NORIAKIIIJIMAet al.
52
HDL was about two to three times higher than those in VLDL and LDL, and thereafter almost all of radioactivity was found in HDL fraction. It was found that the main radioactivity in HDL fraction was TG. The distribution of radioactivities in the lipid classes of various tissues in carp at 72 hr after force-feeding [l-‘4C]dioleoyl PC is shown in Table 1. As shown in the column of lipid content, about 60% of whole body lipids were distributed in other tissues, followed by muscle (16.2%) and skin (8.7%). However, high radioactivity was detected in the intestine (28.5%), gill (25.6%) and other tissues (18.8%) in the form of PC. DISCUSSION
In fish, the digestibility of dietary lipids seems to depend on the environmental water temperature (Leger, 1985; Henderson and Tocher, 1987). At high water temperature (20-25°C) in summer season (July and August), TG hydrolase activity in carp intestinal fluid reached maximum around 9 hr after dosing soybean oil, and the peak of lipid absorption was recognized around 9 and 12 hr after dosing 14C labelled fatty acids (Robinson and Mead, 1973; Kayama and Iijima, 1976; Iijima, Aida and Kayama, unpublished data) or TG (Kayama and Iijima, 1976). On the other hand, at low water temperature (12-15°C) in winter, intraluminal TG hydrolase activity in carp very slowly increased up to 24 hr after force-feeding soybean oil (Fig. l), and maximum radioactivity in blood plasma continued from 28 and 72 hr after dosing [l-14C]triolein (Fig. 2). Moreover, high radioactivity still remains unabsorbed in the intestinal contents even at 72 hr (Table 1). It was assumed from the above results that in winter (November and December), dietary TG was hydrolysed very slowly in the intestinal lumen according to the delay of lipase secretion in the intestinal
lumen. The products, MG and FFA, were mainly reconstituted into TG and PL in the intestinal mucosa (Figs 3 and 4), followed by the release as lipoproteins into the blood circulatory system (Figs 2, 5 and 6). Recently, we separated carp plasma lipoproteins into four fractions, d < 1.006 g/ml, d = 1.006-l .063 g/ml, d = 1.063-1.125 g/ml and d = 1.125-1.21 g/ml, by their density intervals, and these four lipoprotein fractions corresponded to lipoprotein bands: Band IV (d < 1.006 g/ml), Band III (d = 1.006-1.063 g/ml), Band II (d = 1.063-1.125 g/ml) and Band I (d = 1.125-1.21 g/ml), which by PAGE were assumed to be VLDL, intermediate density lipoprotein (IDL, LDL,), LDL, and HDL, respectively. Therefore, as shown in Fig. 2, the lipoprotein bands, Band IV, Band III, Band II and Band I, were tentatively named as VLDL, LDL,, LDL, and HDL, respectively. However, we must further investigate the relationship of carp plasma lipoprotein fractions separated by sequential ultracentrifugation, PAGE and HPGC, as described previously (Hermier et al., 1985; Babin, 1987). When [l-‘4C]triolein was fed to carp, only a little radioactivity was detected in lipoprotein Band IV (VLDL) by PAGE (Fig. 2), and most of the radioactivity was detected in Band III (LDL,), Band II (LDL,) and Band I (HDL). Moreover, from 12 to 72 hr after feeding, high radioactivity was detected in both LDL and HDL fractions (Figs 5 and 6). It was considered from the above results that under the very slow absorption in winter, dietary TG were mainly released into the blood circulatory system in the form of LDL,-, LDL,- and HDL-like particles smaller than VLDL. In mammals, it was recognized that absorbed polyene-PC was incorporated preferentially into the HDL fraction of plasma (Zierenberg et al., 1979; Zierenberg and Grundy, 1982). Although high incorporation of PC into HDL was assumed to be due to Others
::
0
12
20
28
48 Time
72
(h)
Fig. 12. Time course changes of radioactivity distributions in the lipid classes of carp plasma lipoprotein fractions separated by HPGC after force-feeding [I-14C]dioleyl PC. The radioactivities of TG (-_O-), FFA (--O--), MG and DG (--A--) and PL (-¤--) were counted as described in Fig. 6.
767,300
11,903.8
5.16 4.27 1.10 6.60 0.89 5.83 9.31 0.95 0.71 150.19 18.12 2.09 4(mI) 1.2 0.73 123.86 335.01
335.01
Sum
(%)
K; (t8:8) (95.3)
r$ (1.2)
g;
(4.7) (28.5) (1.8) (0.5) (5.7) (0.7) (I.11 (25.6) (Tr.)
0.5 0.1 3.5 0.9 3.0 0.1
LPC 0.5 9.2 0.9 0.3 0.6 5.0 0.5 1.1 2.2 20.6 5.0
1.5
SPM
*Carp used was 348.27 g of body weight. tOther tissues contain skeleton, head, tins, scales and drips. fOthers contain galactolipids, diacylglyceryl ethers, fatty aldehydes and hydrocarbons. Abbreviations are as described in Table I.
36,000 219,000 14,Ofxl 3600 436,000 5200 8200 I%,600 309 100 wIO0 21,800 2200 9000 Uoo 8400 I44,Ooo 13 1,300
Total radioactivity (dpm)
34.7 145.9 153.2 25.6 248.8 109.1 444.3 303.7 5.1 101.3 1930.0 1030.0 92.1 29.3 7.2 13.5 7230.0 I11,869.l
Lipid content (msf
Intestinal content Intestine Kidney Spleen Hepstopancreas Adipose tissue Gonad Gil1 Gall-bladder Brain Muscle Skin Swim-btadder Ptasma Erythrocyte Heart other tissuest whole bcdy
-
Tissue we&h1 (wet) tSf
61.2 58.1 45.8 34.6 64.2 55.9 49.3 42.4 33.5 8.1 47.2 59.6
57.8
PC 63.4 70.2 2.6 3.6 4.8 5.0 4.0 5.0 3.4 5.6 2.3 3.2 3.6
PI+PS 2.2 11.5 4.8 4.7 9.1 2.0 4.6 7.1 1.4 6.6 0.4 6.1 3.0 6.8
PE
DG
2.7 0.5 1.7 8.9 15.5 2.8 0.1 1.4 5.5 4.7 0.3 0.z 2.6 8.8 8.3 6.5 2.2 11.2 3.0 0.4 3.0
-
MG
0.5 3.1 3.5 0.1
-
ALC
3.1 4.2
2.4 1.6 0.6 0.9 2.9 6.2 4.1 10.3 5.5 3.5 10.5
FFA
8.5
9.6 4.3 17.3 34.2 51.2 5.2 30.2 11.8 29.7 38.2 3.3 16.6 12.5
17.7
TG
Distribution of radioactivity in the lipid classes (%)
0.3
0.1 I.1 -
;:: -
G -
CE+ WE
Table 2. Distribution of radioactivity in the lipid classes of various tissues of carp at 72 hr after force-feeding [I-“CJdioleyl phosphatidyIcholine*
3; 4.4
13.2 0.1 6.6 2.0 6.1 5.4 7.9 56.3
8.0 5.8
7.0 2.8 2.6
Others$
54
NORIAKIIUIMAet al.
the selective transfer of CHY-PC (Eisenberg, 1984), direct secretion of nascent HDL by the intestine may also have contributed (Glickman and Magun, 1986). The metabolic fate of dietary PC has been investigated in carp by dosing [l-i4C]dioleyl PC. In winter the absorption of dietary TG proceeded slowly in carp at 72 hr after feeding (Figs 1 and 2); however, dietary PC was more readily absorbed than TG (Fig. 7). Moreover, from the present results, three points can be mentioned about the intestinal digestion and absorption in carp: (a) the radioactivity in the fatty acids of the l-position of plasma PC was approximately two to three times higher than that in a 2-position at the peak of lipid absorption (20 hr) (Fig. 10) (in other words, dietary PC preferentially hydrolysed its 2-acyl ester bond in carp intestinal lumen); (b) dietary PC appeared mainly as TG in blood plasma (Fig. 8); and (c) a high amount of radioactivity (28.5%) was distributed in the intestinal tissues (Table 2). We assumed from the above results that dietary PC, hydrolysed into I-acyl 1ysoPC and FFA by intraluminal phospholipase A,, were absorbed in the intestinal mucosa, and mainly resynthesized into TG and PL, especially PC. Then almost all of TG was released in blood plasma mainly as HDL followed in the decreasing order of LDL and
VLDL (Figs 7, 8 and 11). On the other hand, PC might be metabolized by two pathways that were directly secreted into blood plasma as the main form of HDL (Figs 7, 11 and 12) or incorporated as the constituent of internal membrane in the enterocytes (Table 2). Moreover, we also assumed that a part of I-acyl 1ysoPC was hydrolysed to FFA and glycerylphosphorylcholine, because a major part of dietary PC-fatty acids were incorporated into plasma TG (Fig. 8). Therefore, we now propose from the present results and the previous reports (Kayama and Iijima, 1976; Iijima et al., 1985; Mankura et al., 1987; Iijima, Aida and Kayama, unpublished data) that, in carp, a part of dietary lipids (TG and PC) were secreted both as LDL and VLDL by the intestinal mucosa, but the main transport form of lipids was HDL. In carp, HDL is the predominant class of lipoproteins in the plasma (Iijima et al., 1985; Iijima, Aihara, Kayama, Okazaki and Hara, unpublished data) like other bony fishes (Babin, 1989; Chapman,l980). This feature provides HDL with an important role in the transport of lipids. Leger (1985) proposed that high HDL levels may be due either to a low degradation rate or to a high hepatic synthesis of nascent particles or to a fast degradation of the very low density particles by lipoprotein lipase leading to release of apoprotein constituting the HDL. Moreover, the fact that CHY and VLDL are always very low in the plasma could be in favour of the last two hypotheses. Black and Skinner (1986) investigated the relationship between the concentrations of plasma lipoproteins and the activities of lipoprotein lipase and salt-resistant lipase in rainbow trout, and they suggested that HDL served a role similar to that of VLDL in the transport and uptake of fatty acids for energy production besides its function in the transport of cholesterol and as a reservoir for apoproteins. We now propose that the one reason for high HDL levels is the direct secretion of HDL from the intesti-
nal mucosa in addition to the above aspects. That is, in carp HDL seems to play a central role for the transport of dietary lipids, cholesterol, TG and PL from the enterocytes to various tissues instead of CHY and VLDL in mammals (Green and Glickman, 1981). It was recognized that also in the fishes (Babin, 1989; Black and Skinner, 1986), both lipoprotein lipase and salt-resistant lipase, which correspond to mammalian hepatic triglyceride lipase (Smith and Powell, 1984; Kinunnen, 1984), were present in numerous tissues: red and white muscle, heart, brain, liver, adipose tissue and ovaries, and intestinal lipoproteins were hydrolysed by these lipases. Released free fatty acids were incorporated into various tissues in the same manner as in mammals. It was assumed that also in carp, dietary lipids secreted as HDL, LDL and VLDL in blood plasma were hydrolysed by lipoprotein lipase or hepatic TG lipase (salt-resistant lipase), and then released fatty acids were taken up by various tissues, especially gill, muscle and other tissues (head, bone, fins etc.) mainly as PC and TG (Tables 1 and 2); however, dietary TG and PC were finally deposited as PC rather than TG in the whole body of carp in winter. REFERENCES
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P. K. J. (1984) Hepatic endothelial lipase. In
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C. M. II. (1977) The origin of chylomicron
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