Effect of Complete Parenteral Nutrition Using Fat Emulsion on Liver YOSHIKO KOGA, M.D.,* KEIICHI IKEDA, M.D.,t KIYOSHI INOKUCHI, M.D., D.Sc.t
Four to 8 weeks of complete parenteral nutrition including fat emulsion, 10, Initralipid, from 2 to 4 gm/kg/day, was studied in puppies. The following conclusions were reached: At the end of 4-8 weeks of complete parenteral nutrition using the infusion of fat emulsion, the lipid content of the liver was twice the value in control animals, wlhiclh returned to the approximate normal value in a short period after the interruption of the fat emulsion. This increase in the lipid content was considered to be caused by a proper metabolic process of fat emulsion and would seem to indicate that the daily infusion of the fat emulsion from 2 to 4 gm could be performed safely for an extended period of time.
From the Second Department of Surgery, Kyushu University School of Medicine, Fukuoka, Japan
cording to the fat intake. The experimental animals were divided into three grouips. Grouip 1: two animals (4 week experiment) received 2 gm of fat/kg/day. Grouip 2: three animals (4 week experiment) received 3 gm of fat/kg/day. Grouip 3: three animals (4 week experiment) and two (8 week experiment) received 4 gm of fat/kg/day. Ten per H Yi U1 .x;1XI lvr lHox and complete parenteral nuitrition cent Intralipid° was uised as the fat emulsion (Table 2). It are often uised in pediatric suirgery. For many reasons, was infuised over an 8-10 houir period in each 24 hours with fat emtulsion is necessary in order to obtain the reqtuired in- 8 ui of heparin/kg of body wt/hr. Complete parenteral travenouis suipply of energy in suich cases. This study was nuitrition, with 31 gm of carbohydrate and 4 gm of amino clesigned to determine the effect of the prolonged infuision acid mixttures/kg of body weight was continuied after the 4 of fat emuilsion on the liver of immatuire dogs who were week experiment for 5 days in one animal (dog 36, Grouip 2) maintained on a complete parenteral nuitrition, incluiding and for 10 days in a second animal (dog 44, Grouip 3). The "I ntralipid" for 4 to 8 weeks. litter mate controls (seven animals) were fed Clea dog food (commercial food with an adequiate content of calories, Materials and Methods minerals and vitamines) at the same caloric levels as the exSeven, two or three-member litters of puippies, or a total perimental animals. The average daily fat intake was 3.5 of 17 animals, 8-10 weeks old were uised. A silicone ruibber gm/kg of body weight. Duiring the experiment uising the fat catheter was inserted into an external juiguilar vein and emuilsion, blood samples were taken at 2 week intervals in threaded into the suiperior vena cava of one or two animals both the experimental and the control animals. In dogs 36 of each litter.5 The animals were suistained excluisively by and 44, the blood was drawn after 5 and 10 days respectiveveinl receiving 140 Cal/kg of body wt/day. The soluition, in- ly, on a fat free soluition. The concentrations of plasma fused over a 21-23 houir period daily, consisted of 4 gm of lipids, electrophoretic analysis of plasma by the method of amino acid mixtuires/kg of body weight, carbohydrate, fat Noble,'2 the fatty acid composition of the plasma lipids, and all the vitamins and minerals recommended for growth seruim GOT and GPT, and other various determinations in dogs (Table 1).5 The provision of carbohydrate varied ac- were carried ouit on each blood sample." The animals were sacrificed by exsanguiination, 12-14 houirs after the compleSubhmnitted for publication Juntle 17, 1974. tion of the last fat emuilsion in the experimental animals ex° Inistruictor cluiding dogs 36 and 44, which were sacrificed in the same t Associate Professor and Chief, Division of Pediatric Suirgery. manner after 5 and 10 days respectively on a fat free solut Professor and Chairman. Reprinmt re(qI(csts: Y oshiko Koga, M.D., The 2nd Department of Stirgery. tion. The control animals were sacrificed after overnight Kvmmshbu Universitv School of NMedicine, Ftoktuoka City, Fulkuloka Prefec-
tolre. Japan.
° Blood Plasma Co. of Japan, Japan.
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TABLE 1. Composition of the Intravenous Diet
Ntitrients (/kg/day) Protein 4.0 Carbohydrate 22-31 Fat 2-4 Calories 140 Water 140-190
Vitamins (/kg/day)
gm gm gm Cal ml
A D E Choline B12 Folic Acid Thiamine Riboflavin Pyridoxine
1,000 100 0.51 25 0.003 0.015 5.0 1.0 1.5 2.5 1.0 0.1 0.007 2.0
Pantothenic acid Niacin K Biotin PABA
fast. At the time of necropsy, livers were removed and were fixed in 10% neuitral buffered formalin. Tissue specimens from livers were frozen and stored at - 20 C for biochemical stuidies. Tissue specimens were subjected to variotus histopathological procedures, and those histochemical procedures characterizing "intravenouis fat pigment."" Hepatic lipids were extracted,8 evaporated to dryness and determined as "total lipids" by weight. The lipid fractions were separated by thin-layer chromatography on silica gel G, and the quantitative determinations were performed by the following methods: Lipid phosphorus, method of Bautman.3 Total cholesterol, method of Zturkowski; 15 Triglycerides, method of Fletcher;7 Nonesterified fatty acids (NEFA), method of Duncombe.f6 The fatty acid composition of the hepatic lipids was determined by gas-liquiid chromatography.
Results 1. Liver, Spleen and Lung Weight There were no remarkable changes in the liver, spleen and lung weight, calculated per gram weight.
Triglycerides NEFA
0.11 0.04 0.06
Clea Dog Food (gm/100 gm) 3.35 0.53 0.01 0.02
Lipid phosphorus Cholesterol B. Fatty Acid Composition (wt. %) of Intralipid and of Triglycerides of Intralipid and Clea Dog Food Fatty Triglycerides Triglycerides Acid of Intralipid of Clea dog food Intralipid C16 = 0 C16= 1 C18 = 0 C18 = 1 C18 = 2
Others
11.6 0.4 3.8 26.6 50.0 7.6
11.2 1.1 3.2 27.6 49.4 7.5
20.4 2.5 11.1 36.3 25.2 4.2
I.U. mg mg mg mg mg mg mg mg mg mg mg
2. Histopathological Studies
The experimental animals showed a slight accumuilation of fat in the parenchymatous cells of the liver, and the proliferation of and the accumuilation of fat in reticuloendothelial cells of the liver, spleen and lung. " Intravenous fat pigment" in Kupffer cells and the accumuilation of reticuiloendothelial cells with "intravenous fat pigment" were demonstrated in the liver. No other pathological changes were defined microscopically in the tissue specimens collected from major organs. 3. Hepatic Lipid (a) Total Lipid (Fig. 1). The three experimental animals averaged twofold increases in the total lipid content. In dogs 36 and 44, the total lipid content returned to approximately the control value. m- 50 I
LL.
LU
40
T,%iu.il: 2. A. Lipid Composition of Intralipid and Clea Dog Food Intralipid (gm/100 ml) 9.79
I.U. I. U.
Minerals (mg/kg/day) 72 Caleiuim Phosphorus 96 120 Sodiuim 170 Potassiuim Chloride 315 Magnesium 20 Copper 0.1 Cobalt 0.05 Manganese 0.2 Zinc 0.2 Iodine 0.06 Iron 1.0
x x
0
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I
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2
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0 L a
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A
B
Fw;. 1. Effect of complete parenteral nutrition including fat emulsion on the extracted lipid of the liver (total lipid). "A" represents the result of the 8-week experiment. "B" represents the results of the 4-week experiment.@ Grouip 1. 0 Grotup 2. X Group 3. .j-fl-j4 Control group (the mean valuie and the range of the value).
Ann.
KOGA, IKEDA AND INOKUCHI
188 I.0o
10.0
-
There was remarkable similarity in the fatty acid composition of the hepatic triglyceride fraction of the 4 and 8
x
week experimental animals, that of triglyceride component of the fat emuilsion and of the fat emuilsion as a whole. Dogs 36 and 44, differed in their patterns of acids from other experimental animals.
x
I
wV 5.0
Stirg. Febriiary 1975
O.S
Plasma Lipids"' Two to threefold increases in the plasma lipid phosphoruis, cholesterol and triglyceride levels were detected throuighouit the 4-8 weeks of the infusion of fat emuilsion and those increased plasma lipid fractions were proven to be of endogenotus origin. After 5 and 10 days on a fat-free soluition, the levels of plasma lipid fractions of dogs 36 and 44 decreased to approximately the initial values. The fatty acid composition of plasma lipid fractions was similar to that of hepatic lipid fractions. Lipoprotein elecand trophoresis on agarose gel disclosed that lipoproteins increased in the experimental animals during the 4 and 8 weeks of the infuision of the fat emulsion. 5. Serum GOT and GPT There was no significant deviation from the normal in seruim GOT and GPT. Slight ketonemias were observed duiring the 4 and 8 weeks of the infuision of the fat emuilsion. There were no remarkable changes in blood sugar, venous blood pH or in other variotus determinations. The animals were in good condition. In both the experimental and the control animals, their growth, development and weight gains were observed to be approximately normal. 4.
CHOLESTEROL TRIGLYCERIDES
NEFA
LIPID PHOSPHORUS
F.(;. 2. Effect of complete parenteral ntitrition inclutding fat emlulsion on the hepatic lipid fractions in the 8-week experiment. X Grouip 3.---
Control Group.
(b) Hepatic Lipid Fractions (Figs. 2 and 3). In the 4 and 8 week experimental animals there was a marked increase in hepatic lipid fractions. There were twofold increases in the lipid phosphoruis, cholesterol and NEFA contents and a slight increase in the triglyceride content. In dogs 36 and 44, the hepatic cholesterol content was below the control valuie, and the lipid phosphoruis and NEFA contents were
approximately the control valuie. The triglyceride content slightly higher than that of the control group.
was
(c) Fatty Acid Composition of the Hepatic Lipid Fractions (Fig. 4). In the
experimental animals, the fatty acid
composition of hepatic phospholipids differed from that of the fat emuilsion and of its phospholipid component. In the 4 and 8 week experimental animals, the fatty acid composition of the cholesterol esters differed from that of the fat emtulsion, and of dogs 36 and 44. However, it had
considerable similarity to the fatty acid composition of the fat emuilsion as a whole.
a
f-
Discussion Becauise Intralipid consists of triglycerides, phospholipids, cholesterol and NEFA, it would seemn important to investigate whether the increased hepatic fractions
10.0
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CHOLESTEROL
TRIGLYCERIDES
NEFA
LIPID PHOSPHORUS
Fu.. 3. Effect of complete parenteral ntutrition inclhiding fat emuilsion on the hepatic lipid fractions in the 4-week experiment. Grouip 1. 0 Grouip 2. X Control Grouip 3. 1igrotup (the mean valtue and the range of the valtue).
.
Vol. 181 * No. 2
50. 400 30. 2010.
COMPLETE PARENTERAL NUTRITION PHOSPHOLI PIDS
X
2!WII I I IIIIIII )(o0
X
o
".
xo
x
-5
5 4
3.
CHOLESTEROL ESTERS
3
-e---- -
o
>-
*
*
_e
TRIGLYCERIDES I
II
-pl
2 0.
4, Ar.raillill willioll
IIEE I MdI
I
A
c16=0
B
A
c16=I
B
A
B
c18=0
A
c18=I
B
A
B
c18=2
4. Changes in the fatty acid composition (wt. % ) of liver phospholipids, cholesterol esters and triglycerides in the 4 to 8-week complete parenteral ntotrition. "A' represents the restilt of the 8-week experiment. "B' represents the resuilts of the 4-week experiment and of dogs 36 and 44 after 5 and 10 days, respectively, on a fat-free soltution after 4 weeks. O dog 36. 0 dog 44. -H-I-f-HControl grouip (the mean value and the range Fo..
of the valtue).----- Intralipid.
were of exogenouis origin, i.e., they were the accumuilation of the fat emuilsion in the liver, or were of endogenous origin. To determine this, the fatty acid composition of the hepatic lipid fractions were examined. Because of the extremely short half life of NEFA,14 the NEFA of the infused fat emuilsion might be metabolized rapidly and those in the liver were assuimed to be endogenous. It was found that the fatty acid composition of phospholipid component of the fat emtulsion and the hepatic lipids were dissimilar as were those of cholesterol esters in the fat emulsion and the hepatic lipids. The fatty acid composition of the hepatic triglycerides had striking similarity to that of the fat emulsion as a whole. In this experiment, the plasma triglycerides whose fatty acid composition was similar to that of the fat emuilsion as a whole were proven to be endogenous." There is experimental evidence that the acid mixtuires of the triglycerides were altered by the dietary fats.' Therefore, in the case of the intravenouis fat administration, the same phenomenon might apply to the hepatic triglycerides. Thuis, the evidence has been obtained that the increased hepatic lipid fractions might come from endogenous synthesis.
In this experiment, the three experimental animals averaged twofold increases in the total hepatic lipids and in hepatic lipid fractions, regardless of the length of the total infuision period and the daily volume of the fat emulsion administered. Those changes in the hepatic lipids, however, diminished and returned to nearly the initial values shortly after the interruiption of the fat emulsion as noted in dogs 36 and 44. It is worthy of note that the long-term daily fat emulsion, largely composed of triglycerides, led to marked rises in the hepatic phospholipid and cholesterol contents as well as in the triglyceride content; and the increases in the former were more prominent. The constituent of the fat emulsion might be reformed into other esters within the liver and reenter the blood stream as lipoproteins. In this experiment, a and A-lipoproteins, reportedly rich in cholesterol and phospholipids,10 seemed to perform the major lipid transport tasks in the experimental animals. What is actually responsible for the increase in the hepatic lipids, partictilarly with phospholipids and cholesterol is not completely uinderstood; however, enhanced endogenous synthesis of a and f-lipoproteins in the liver, appears to have an important role. Evidence for this view is based on our observations that in the 4 and 8 week experimental animals, plasma lipid fractions, proved to be largely endogenous, were also elevated, concomitant with the rise in a and dlipoproteins in the circuilation.11 The infuised fat emulsion might lead to the enhancement in lipid transport tasks for final uptake by the utilizing tissues. a and 0-lipoproteins seemed to be produced vigorously for this purpose, and the cholesterol and phospholipids might be demanded as structural components of lipoproteins.9 Because there is abundant evidence that the liver is the chief source of the circtulating lipoproteins and lipids derived from endogenous synthesis,2 vigorouis production of a and d-lipoproteins might cauise the enhanced production of and the increase in phospholipids and cholesterol in the liver. Although in this experiment the experimental and the control animals received approximately the same amount of fat, the lipid content of the liver was markedly increased when the experimental animals were compared with the control animals. This might be due to the differences in the metabolic pathway between the fat given orally and intravenouisly.4 Five and 10 days after the completion of the last fat emuilsion, however, those changes in the hepatic lipids retturned to approximately the control values and the fatty acid composition of the hepatic cholesterol esters and triglycerides showed different results from those obtained at the end of 4 and 8 weeks of infuision of the fat emulsion. The phospholipids appeared to be influienced little in their fatty acid composition by the fat emulsion, as in the case of dietary fat.' These phenomenon were presumed to indicate that the enhanced lipid metabolism, because of the fat emuilsion, retturned approximately to its initial level in 5-10
190
KOGA, IKEDA AND INOKUCHI
days after its interruption, exerting no residual ill effect. Fuirthermore, it was obtained that the animals remained in good condition, and their growth, development, and weight gain were observed to be approximately normal. Thus, daily infusion of fat emulsion, from 2 to 4 gm/kg of body weight couild be performed safely for an extended period. References 1. Ahrens, E. H., Jr., Instill, W., Jr., Hirsh, J., et al.: The Effect on Huiman Sertimlipids of a Dietary Fat, Highly Unsaturated, but Poor in Essential Fatty Acids. Lancet, 1:115, 1959. 2. Baker, N., Garfinekel, A. S. and Schotz, M. C.: Hepatic Triglyceride Secretion in Relation to Lipogenesis and Free Fatty Acid Mobilization in Fasted and Glucoserefed Rats. J. Lipid Res., 9:1, 1968. 3. Batiman, E. J.: On the Estimation of Organic Phosphoruis. J. Biol. Chem., 59:667, 1924. 4. Cornell, R. P. and Seba, T. M.: Vascular Clearance and Metabolism of Lipid by the Reticuloendothelial System in Dogs. Am. J. Physiol., 221:1511, 1971. 5. Duidrick, S. J., Wilmore, D. S., Vars, H. M. and Rhoads, J. E.: Longterm Total Parenteral Nutrition with Growth, Development and Positive Nitrogen Balance. Surgery, 64:134, 1968.
Ann.
Stirg. Febriiary 1975 -
6. Duincombe, W. G.: The Colorimetric Micro-determination of Longchain Fatty Acids. Biochem. J., 88:7, 1963. 7. Fletcher, M. J.: A Colorimetric Method for Estimating Serum Triglycerides. Clinica. Chim. Acta., 22:393, 1968. 8. Folch, J., Lees, M., Sloane and Stanley, G. H.: A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J. Biol. Chem., 226:497, 1957. 9. Fredrickson, D. S., Levy, R. I. and Lees, R. S.: Medical Progress Fat Transport in Lipoprotein-An Integrated Approach to Mechanisms and Disorders. N. Engl. J. Med., 276:32, 1967. 10. Hillyard, L. A., Entenman, C. C. and Reinhardt, W. O.: Composition and Concentration of Lymph and Serum Lipoproteins During Fat and Cholesterol Absorption in the Dog. J. Biol. Chem., 233:838, 1958. 11. Koga, Y., Ikeda, K. and Inokuchi, K.: Effect of Complete Parenteral Nuitrition Incluiding Fat Emulsion on Plasma Lipid. (In press) 12. Noble, R. P.: Electrophoretic Separation of Plasma Lipoprotein in Agarose Gel. J. Lipid Res., 9:693, 1968. 13. Thompson, S. W., Johnson, F. B. and Forbes, A. L.: Staining Characteristics of a Pigment Associated with Intravenous Fat Alimentation. Lab. Invest., 7:533, 1958. 14. Yoshitoshi, Y., Naito, C., Okaniwa, H., et al.: Kinetic Stuidies on Metabolism of Lipoprotein Lipase. J. Clin. Invest., 42:707, 1963. 15. Zuirkowski, O.: A Rapid Method for Cholesterol Determination. Clin. Chem., 10:451, 1964.
Four to 8 weeks of complete parenteral nutrition including fat emulsion, 10, Initralipid, from 2 to 4 gm/kg/day, was studied in puppies. The following conclusions were reached: At the end of 4-8 weeks of complete parenteral nutrition using the infusion of fat emulsion, the lipid content of the liver was twice the value in control animals, wlhiclh returned to the approximate normal value in a short period after the interruption of the fat emulsion. This increase in the lipid content was considered to be caused by a proper metabolic process of fat emulsion and would seem to indicate that the daily infusion of the fat emulsion from 2 to 4 gm could be performed safely for an extended period of time.
From the Second Department of Surgery, Kyushu University School of Medicine, Fukuoka, Japan
cording to the fat intake. The experimental animals were divided into three grouips. Grouip 1: two animals (4 week experiment) received 2 gm of fat/kg/day. Grouip 2: three animals (4 week experiment) received 3 gm of fat/kg/day. Grouip 3: three animals (4 week experiment) and two (8 week experiment) received 4 gm of fat/kg/day. Ten per H Yi U1 .x;1XI lvr lHox and complete parenteral nuitrition cent Intralipid° was uised as the fat emulsion (Table 2). It are often uised in pediatric suirgery. For many reasons, was infuised over an 8-10 houir period in each 24 hours with fat emtulsion is necessary in order to obtain the reqtuired in- 8 ui of heparin/kg of body wt/hr. Complete parenteral travenouis suipply of energy in suich cases. This study was nuitrition, with 31 gm of carbohydrate and 4 gm of amino clesigned to determine the effect of the prolonged infuision acid mixttures/kg of body weight was continuied after the 4 of fat emuilsion on the liver of immatuire dogs who were week experiment for 5 days in one animal (dog 36, Grouip 2) maintained on a complete parenteral nuitrition, incluiding and for 10 days in a second animal (dog 44, Grouip 3). The "I ntralipid" for 4 to 8 weeks. litter mate controls (seven animals) were fed Clea dog food (commercial food with an adequiate content of calories, Materials and Methods minerals and vitamines) at the same caloric levels as the exSeven, two or three-member litters of puippies, or a total perimental animals. The average daily fat intake was 3.5 of 17 animals, 8-10 weeks old were uised. A silicone ruibber gm/kg of body weight. Duiring the experiment uising the fat catheter was inserted into an external juiguilar vein and emuilsion, blood samples were taken at 2 week intervals in threaded into the suiperior vena cava of one or two animals both the experimental and the control animals. In dogs 36 of each litter.5 The animals were suistained excluisively by and 44, the blood was drawn after 5 and 10 days respectiveveinl receiving 140 Cal/kg of body wt/day. The soluition, in- ly, on a fat free soluition. The concentrations of plasma fused over a 21-23 houir period daily, consisted of 4 gm of lipids, electrophoretic analysis of plasma by the method of amino acid mixtuires/kg of body weight, carbohydrate, fat Noble,'2 the fatty acid composition of the plasma lipids, and all the vitamins and minerals recommended for growth seruim GOT and GPT, and other various determinations in dogs (Table 1).5 The provision of carbohydrate varied ac- were carried ouit on each blood sample." The animals were sacrificed by exsanguiination, 12-14 houirs after the compleSubhmnitted for publication Juntle 17, 1974. tion of the last fat emuilsion in the experimental animals ex° Inistruictor cluiding dogs 36 and 44, which were sacrificed in the same t Associate Professor and Chief, Division of Pediatric Suirgery. manner after 5 and 10 days respectively on a fat free solut Professor and Chairman. Reprinmt re(qI(csts: Y oshiko Koga, M.D., The 2nd Department of Stirgery. tion. The control animals were sacrificed after overnight Kvmmshbu Universitv School of NMedicine, Ftoktuoka City, Fulkuloka Prefec-
tolre. Japan.
° Blood Plasma Co. of Japan, Japan.
186
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187
TABLE 1. Composition of the Intravenous Diet
Ntitrients (/kg/day) Protein 4.0 Carbohydrate 22-31 Fat 2-4 Calories 140 Water 140-190
Vitamins (/kg/day)
gm gm gm Cal ml
A D E Choline B12 Folic Acid Thiamine Riboflavin Pyridoxine
1,000 100 0.51 25 0.003 0.015 5.0 1.0 1.5 2.5 1.0 0.1 0.007 2.0
Pantothenic acid Niacin K Biotin PABA
fast. At the time of necropsy, livers were removed and were fixed in 10% neuitral buffered formalin. Tissue specimens from livers were frozen and stored at - 20 C for biochemical stuidies. Tissue specimens were subjected to variotus histopathological procedures, and those histochemical procedures characterizing "intravenouis fat pigment."" Hepatic lipids were extracted,8 evaporated to dryness and determined as "total lipids" by weight. The lipid fractions were separated by thin-layer chromatography on silica gel G, and the quantitative determinations were performed by the following methods: Lipid phosphorus, method of Bautman.3 Total cholesterol, method of Zturkowski; 15 Triglycerides, method of Fletcher;7 Nonesterified fatty acids (NEFA), method of Duncombe.f6 The fatty acid composition of the hepatic lipids was determined by gas-liquiid chromatography.
Results 1. Liver, Spleen and Lung Weight There were no remarkable changes in the liver, spleen and lung weight, calculated per gram weight.
Triglycerides NEFA
0.11 0.04 0.06
Clea Dog Food (gm/100 gm) 3.35 0.53 0.01 0.02
Lipid phosphorus Cholesterol B. Fatty Acid Composition (wt. %) of Intralipid and of Triglycerides of Intralipid and Clea Dog Food Fatty Triglycerides Triglycerides Acid of Intralipid of Clea dog food Intralipid C16 = 0 C16= 1 C18 = 0 C18 = 1 C18 = 2
Others
11.6 0.4 3.8 26.6 50.0 7.6
11.2 1.1 3.2 27.6 49.4 7.5
20.4 2.5 11.1 36.3 25.2 4.2
I.U. mg mg mg mg mg mg mg mg mg mg mg
2. Histopathological Studies
The experimental animals showed a slight accumuilation of fat in the parenchymatous cells of the liver, and the proliferation of and the accumuilation of fat in reticuloendothelial cells of the liver, spleen and lung. " Intravenous fat pigment" in Kupffer cells and the accumuilation of reticuiloendothelial cells with "intravenous fat pigment" were demonstrated in the liver. No other pathological changes were defined microscopically in the tissue specimens collected from major organs. 3. Hepatic Lipid (a) Total Lipid (Fig. 1). The three experimental animals averaged twofold increases in the total lipid content. In dogs 36 and 44, the total lipid content returned to approximately the control value. m- 50 I
LL.
LU
40
T,%iu.il: 2. A. Lipid Composition of Intralipid and Clea Dog Food Intralipid (gm/100 ml) 9.79
I.U. I. U.
Minerals (mg/kg/day) 72 Caleiuim Phosphorus 96 120 Sodiuim 170 Potassiuim Chloride 315 Magnesium 20 Copper 0.1 Cobalt 0.05 Manganese 0.2 Zinc 0.2 Iodine 0.06 Iron 1.0
x x
0
ox
X
30
DOG 36
u
I-
I
2 I~~~ IIIIISII ~~
2
DOG 44
0 L a
10
10U
.l
x
LU
A
B
Fw;. 1. Effect of complete parenteral nutrition including fat emulsion on the extracted lipid of the liver (total lipid). "A" represents the result of the 8-week experiment. "B" represents the results of the 4-week experiment.@ Grouip 1. 0 Grotup 2. X Group 3. .j-fl-j4 Control group (the mean valuie and the range of the value).
Ann.
KOGA, IKEDA AND INOKUCHI
188 I.0o
10.0
-
There was remarkable similarity in the fatty acid composition of the hepatic triglyceride fraction of the 4 and 8
x
week experimental animals, that of triglyceride component of the fat emuilsion and of the fat emuilsion as a whole. Dogs 36 and 44, differed in their patterns of acids from other experimental animals.
x
I
wV 5.0
Stirg. Febriiary 1975
O.S
Plasma Lipids"' Two to threefold increases in the plasma lipid phosphoruis, cholesterol and triglyceride levels were detected throuighouit the 4-8 weeks of the infusion of fat emuilsion and those increased plasma lipid fractions were proven to be of endogenotus origin. After 5 and 10 days on a fat-free soluition, the levels of plasma lipid fractions of dogs 36 and 44 decreased to approximately the initial values. The fatty acid composition of plasma lipid fractions was similar to that of hepatic lipid fractions. Lipoprotein elecand trophoresis on agarose gel disclosed that lipoproteins increased in the experimental animals during the 4 and 8 weeks of the infuision of the fat emulsion. 5. Serum GOT and GPT There was no significant deviation from the normal in seruim GOT and GPT. Slight ketonemias were observed duiring the 4 and 8 weeks of the infuision of the fat emuilsion. There were no remarkable changes in blood sugar, venous blood pH or in other variotus determinations. The animals were in good condition. In both the experimental and the control animals, their growth, development and weight gains were observed to be approximately normal. 4.
CHOLESTEROL TRIGLYCERIDES
NEFA
LIPID PHOSPHORUS
F.(;. 2. Effect of complete parenteral ntitrition inclutding fat emlulsion on the hepatic lipid fractions in the 8-week experiment. X Grouip 3.---
Control Group.
(b) Hepatic Lipid Fractions (Figs. 2 and 3). In the 4 and 8 week experimental animals there was a marked increase in hepatic lipid fractions. There were twofold increases in the lipid phosphoruis, cholesterol and NEFA contents and a slight increase in the triglyceride content. In dogs 36 and 44, the hepatic cholesterol content was below the control valuie, and the lipid phosphoruis and NEFA contents were
approximately the control valuie. The triglyceride content slightly higher than that of the control group.
was
(c) Fatty Acid Composition of the Hepatic Lipid Fractions (Fig. 4). In the
experimental animals, the fatty acid
composition of hepatic phospholipids differed from that of the fat emuilsion and of its phospholipid component. In the 4 and 8 week experimental animals, the fatty acid composition of the cholesterol esters differed from that of the fat emtulsion, and of dogs 36 and 44. However, it had
considerable similarity to the fatty acid composition of the fat emuilsion as a whole.
a
f-
Discussion Becauise Intralipid consists of triglycerides, phospholipids, cholesterol and NEFA, it would seemn important to investigate whether the increased hepatic fractions
10.0
I .o
0 I-
44 U.
5.
~~~~~~e
S
0
:j 0.5'-
0
I
DOG 36 DOG 44
DOG 36 DOG 44
DOG 36
keDOG _44
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+I'hi. . '1 I
11 1
.Jliff _IIIIIIII . ali 1ffitU)
.W
CHOLESTEROL
TRIGLYCERIDES
NEFA
LIPID PHOSPHORUS
Fu.. 3. Effect of complete parenteral ntutrition inclhiding fat emuilsion on the hepatic lipid fractions in the 4-week experiment. Grouip 1. 0 Grouip 2. X Control Grouip 3. 1igrotup (the mean valtue and the range of the valtue).
.
Vol. 181 * No. 2
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COMPLETE PARENTERAL NUTRITION PHOSPHOLI PIDS
X
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4. Changes in the fatty acid composition (wt. % ) of liver phospholipids, cholesterol esters and triglycerides in the 4 to 8-week complete parenteral ntotrition. "A' represents the restilt of the 8-week experiment. "B' represents the resuilts of the 4-week experiment and of dogs 36 and 44 after 5 and 10 days, respectively, on a fat-free soltution after 4 weeks. O dog 36. 0 dog 44. -H-I-f-HControl grouip (the mean value and the range Fo..
of the valtue).----- Intralipid.
were of exogenouis origin, i.e., they were the accumuilation of the fat emuilsion in the liver, or were of endogenous origin. To determine this, the fatty acid composition of the hepatic lipid fractions were examined. Because of the extremely short half life of NEFA,14 the NEFA of the infused fat emuilsion might be metabolized rapidly and those in the liver were assuimed to be endogenous. It was found that the fatty acid composition of phospholipid component of the fat emtulsion and the hepatic lipids were dissimilar as were those of cholesterol esters in the fat emulsion and the hepatic lipids. The fatty acid composition of the hepatic triglycerides had striking similarity to that of the fat emulsion as a whole. In this experiment, the plasma triglycerides whose fatty acid composition was similar to that of the fat emuilsion as a whole were proven to be endogenous." There is experimental evidence that the acid mixtuires of the triglycerides were altered by the dietary fats.' Therefore, in the case of the intravenouis fat administration, the same phenomenon might apply to the hepatic triglycerides. Thuis, the evidence has been obtained that the increased hepatic lipid fractions might come from endogenous synthesis.
In this experiment, the three experimental animals averaged twofold increases in the total hepatic lipids and in hepatic lipid fractions, regardless of the length of the total infuision period and the daily volume of the fat emulsion administered. Those changes in the hepatic lipids, however, diminished and returned to nearly the initial values shortly after the interruiption of the fat emulsion as noted in dogs 36 and 44. It is worthy of note that the long-term daily fat emulsion, largely composed of triglycerides, led to marked rises in the hepatic phospholipid and cholesterol contents as well as in the triglyceride content; and the increases in the former were more prominent. The constituent of the fat emulsion might be reformed into other esters within the liver and reenter the blood stream as lipoproteins. In this experiment, a and A-lipoproteins, reportedly rich in cholesterol and phospholipids,10 seemed to perform the major lipid transport tasks in the experimental animals. What is actually responsible for the increase in the hepatic lipids, partictilarly with phospholipids and cholesterol is not completely uinderstood; however, enhanced endogenous synthesis of a and f-lipoproteins in the liver, appears to have an important role. Evidence for this view is based on our observations that in the 4 and 8 week experimental animals, plasma lipid fractions, proved to be largely endogenous, were also elevated, concomitant with the rise in a and dlipoproteins in the circuilation.11 The infuised fat emulsion might lead to the enhancement in lipid transport tasks for final uptake by the utilizing tissues. a and 0-lipoproteins seemed to be produced vigorously for this purpose, and the cholesterol and phospholipids might be demanded as structural components of lipoproteins.9 Because there is abundant evidence that the liver is the chief source of the circtulating lipoproteins and lipids derived from endogenous synthesis,2 vigorouis production of a and d-lipoproteins might cauise the enhanced production of and the increase in phospholipids and cholesterol in the liver. Although in this experiment the experimental and the control animals received approximately the same amount of fat, the lipid content of the liver was markedly increased when the experimental animals were compared with the control animals. This might be due to the differences in the metabolic pathway between the fat given orally and intravenouisly.4 Five and 10 days after the completion of the last fat emuilsion, however, those changes in the hepatic lipids retturned to approximately the control values and the fatty acid composition of the hepatic cholesterol esters and triglycerides showed different results from those obtained at the end of 4 and 8 weeks of infuision of the fat emulsion. The phospholipids appeared to be influienced little in their fatty acid composition by the fat emulsion, as in the case of dietary fat.' These phenomenon were presumed to indicate that the enhanced lipid metabolism, because of the fat emuilsion, retturned approximately to its initial level in 5-10
190
KOGA, IKEDA AND INOKUCHI
days after its interruption, exerting no residual ill effect. Fuirthermore, it was obtained that the animals remained in good condition, and their growth, development, and weight gain were observed to be approximately normal. Thus, daily infusion of fat emulsion, from 2 to 4 gm/kg of body weight couild be performed safely for an extended period. References 1. Ahrens, E. H., Jr., Instill, W., Jr., Hirsh, J., et al.: The Effect on Huiman Sertimlipids of a Dietary Fat, Highly Unsaturated, but Poor in Essential Fatty Acids. Lancet, 1:115, 1959. 2. Baker, N., Garfinekel, A. S. and Schotz, M. C.: Hepatic Triglyceride Secretion in Relation to Lipogenesis and Free Fatty Acid Mobilization in Fasted and Glucoserefed Rats. J. Lipid Res., 9:1, 1968. 3. Batiman, E. J.: On the Estimation of Organic Phosphoruis. J. Biol. Chem., 59:667, 1924. 4. Cornell, R. P. and Seba, T. M.: Vascular Clearance and Metabolism of Lipid by the Reticuloendothelial System in Dogs. Am. J. Physiol., 221:1511, 1971. 5. Duidrick, S. J., Wilmore, D. S., Vars, H. M. and Rhoads, J. E.: Longterm Total Parenteral Nutrition with Growth, Development and Positive Nitrogen Balance. Surgery, 64:134, 1968.
Ann.
Stirg. Febriiary 1975 -
6. Duincombe, W. G.: The Colorimetric Micro-determination of Longchain Fatty Acids. Biochem. J., 88:7, 1963. 7. Fletcher, M. J.: A Colorimetric Method for Estimating Serum Triglycerides. Clinica. Chim. Acta., 22:393, 1968. 8. Folch, J., Lees, M., Sloane and Stanley, G. H.: A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J. Biol. Chem., 226:497, 1957. 9. Fredrickson, D. S., Levy, R. I. and Lees, R. S.: Medical Progress Fat Transport in Lipoprotein-An Integrated Approach to Mechanisms and Disorders. N. Engl. J. Med., 276:32, 1967. 10. Hillyard, L. A., Entenman, C. C. and Reinhardt, W. O.: Composition and Concentration of Lymph and Serum Lipoproteins During Fat and Cholesterol Absorption in the Dog. J. Biol. Chem., 233:838, 1958. 11. Koga, Y., Ikeda, K. and Inokuchi, K.: Effect of Complete Parenteral Nuitrition Incluiding Fat Emulsion on Plasma Lipid. (In press) 12. Noble, R. P.: Electrophoretic Separation of Plasma Lipoprotein in Agarose Gel. J. Lipid Res., 9:693, 1968. 13. Thompson, S. W., Johnson, F. B. and Forbes, A. L.: Staining Characteristics of a Pigment Associated with Intravenous Fat Alimentation. Lab. Invest., 7:533, 1958. 14. Yoshitoshi, Y., Naito, C., Okaniwa, H., et al.: Kinetic Stuidies on Metabolism of Lipoprotein Lipase. J. Clin. Invest., 42:707, 1963. 15. Zuirkowski, O.: A Rapid Method for Cholesterol Determination. Clin. Chem., 10:451, 1964.
