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Effect of intralipid® overdose on brain lipid composition in the adult rabbit and its relation with the route of administration
NutritionResearch,Vol.15,No. 6, pp. 881-888.1995 Copyright0 1995ElsevierScienceLtd Printedin the USA. All rightsreserved 0271-5317/95$9.50+ .OO
Pergamon
EFFECT
OF INTRALIPIDB OVERDOSE ON BRAIN LIPID COMPOSITION IN THE ADULT RABBIT AND ITS RELATION WITH THE ROUTE OF ADMINISTRATION
MD, PhD’ ; JM Moran Pence, MD, PhDI; J. Salas Martinez, MD, PhD’; G. Mahedero Ruiz, MD, PhDl; G. Cruz Villal6n, ChDI; V Climent Mata, MD, PhD*; M. Molina FernAndez’, PhD. ‘Dep. Surgery (chairman: Prof LM Vinagre), *Dep Morphologic Sciences and 13Dep of Bioestatistics. Universidad de Extremadura, Badajoz. Spain. E. Macia Botejara,
ABSTRACT A group of 33 New Zeland rabbits were injected with 4g/kg weight/day of Intralipid (ITL) 20%. They were injected intravenously, intraperitoneally and intragrasticaily. The aim was to study the variations in plasma and brain lipids after fats overdosages. A control group, which was operated without receiving ITL, was also established. After IS days of daily lntralipid dosage, complex lipids and fatty acids (FA) administered with ITL rose in plasma levels in the intravenous and intraperitoneal groups. The study of brain lipids showed decreasing levels of phosphatidilethanolamine (PhE), phosphatidilcholine and phosphatidilserine (PhC+PhS), and palmitoil cerebroside (PC) in those groups parenterally supplemented with ITL, without any variation in the polar lipds/neutral lipids ratio (PUNL). Those groups supplemented with ITL showed increasing levels of the stearic (C-18) and oleic (C-18:1) acids in brain, whereas linoleic (C-18:2) and arachinodic (C-20:4) acids levels decreased. In conclusion, parenteral lntralipid overdosages cause longer -lasting changes in plasma lipids after intraperitoneal administration. A decreasing level of structural lipids, linoleic and arachidonic acids and also the ratio between both is noticed in the brain affecting all supplemented groups. All these changes in the adult animals are different to the ones described in other animals and lactant humans. Key words: Brain lipids; Lipids clearance;
lntraperitoneal
absorption;
Lipid overdose
INTRODUCTION Lipids are the essential constituents of the brain.They may become up to a 60% of the organ weight in dry conditions and they are mainly part of the membranous structures. Polyunsaturated fatty acids (PIJFAs) stand out among the brain fatty acids. They derivate from the linoleic (C-18:2) and the linolenic (C-183) acids. Their elongation and desaturation is mediated by the same enzyme systems and a competition between both is possible. This circumstance allows the brain lipids composition, and as a consequence, the neurological functions and the learning capacity, to be influenced by external contributions of fat, as it has been demonstrated in different studies (l)(2)(3)(4). Changes in brain lipid composition in lactant and premature babies, have been also demonstrated, when following the administration of total parenteral nutrition (TPN) with Intralipid (ITL), rich in lecithins and palmitic (C- 16), oleic (C- 18: 1). linoleic (C- l8:2) and arachidonic (C-20:4) acids (5). However. there are few works which, when studiying the possible changes in lipid body composition after TPN, differentiate the efects that they are due to the composition of the nutritional formula. to the access route of the Iipids(oral. parenteral), whether they are produced with dosage-dependent relation- after an overload and whether these possible changes once the brain is developed. Our aim in this work was to study the lipid composition of the general plasma pool and the brain after overdosage with ITL using different vias (enteral, intraperitoneal and intravenous) in a group of adult mammals.
’ Adress correspondence to: Enrique Macia Botejara. Department of Surgery. Facultad de Medicina. UEX. Av de Elvas s/n. 06071 BADAJOZ. Spain. This work was support for a grant from the Fondo de lnvestigaciones Sanitarias de la Seguridad Social (FISS) no 89/0867 and 91/0147. 881
E.M. BOTEJARA et al.
882
METHODS
AND
MATERIALS
Thirty-three New Zeland rabbits aged 6+1 months housed under the following conditions were used: 14-10 h. cycles of light/darkness, temperature from 20+ to 2” C and 13 renewals of air per hour. All through the experimental period, the animals received water and chow “ad libitum”. The composition of the diet was as follows: Proteins, 15%; Fat, 2,3%; Cellulose 17%; Ash 12%; Starch, 14% and Minerals 2,45%. The whole group was anaesthetised with Ketamina (KetolairO.Lab.Parke-Davis.Morris-Plains, N.J. USA), at doses of 25 mg/kg and preanaesthesia with Fenotiacina (CombelenB.Lab. Bayer, Germany) at doses of 1 ml/Kg. both intramuscularly administered. Experimental groups: Intraneritoneal Group (IP). This group was formed with 9 animals which underwent a minilaparotomy in the right hypocondrium to set a silastic catheter (NutricathB No. 18. Lab. Vigon) in the abde mini1 cavity which’reached the interscapular region and through which ITL at 26% was injected (Kabi Vitrum. Stockholm. Sweden)at a dose of 4 gr. of lipids/Kg/day, for 15 days and in two bolus (at 9.00 a.m. and 20.00 p.m.). Over ten centimeters of catheter were left inside the abdomen, and several drainage paths were opened in its five dystal centimeters. Intravenous Group (IV). This group was formed with 8 animals, to which a silastic catheter (NutricathO No 18. Lab. Vigon) was implanted in the external jugular vein and it was tunnelised to reach the interscapular region. The lipids administration was carried out under the same conditions and using the same doses alreadv described above for erouD IP. IntraeraGic Group GIG). This g&up was formed with 5 an:mals which received the lipids by means of a nasogastric tube Fr 5 made of noliurethane. The dailv amount of lipids received bv each animal was the same and the periods of time were also the same as the one ised in the other groups, but with a single bolus. A simulated surgical operation was practised before treatment started. Control Group (CC). This group was composed of 8 animals. They didn’t received ITL supplement, though they were operated under general anaesthesia to set an inserted catheter. All animals involved in the experiment were weighed at the beginning and at the end of the experiment apart from the clinical daily follow-up and to determine the glucose, urea and creatinine in blood every 48 hours. A 1Oc.c. extraction of blood from the central arteria of the ear was perfommed on the 16th day, 24 hours after the last infusion. Inmediately afterwards, the animals were anaesthetised and a medial laparotomy was carried out. A biobsy of the anterior edge of the major hepatic lobe, diaphragm and intestine was taken. After a medial craneotomy was performed, the encephalic mass and the cerebellum were extracted and maintained at -80°C until it was analysed. This analysis was to be done between the 48 and 72 following hours. Using a Hitachi 705 (Hitachi Ltd. Osaka, Japan) autoanalyser, the levels of Glucose, Urea, Total Cholesterol (TCh), Triglycerides (TG), Phospholipids (PL), total and direct Bilirubin, Aspartatetransferase (AST), Alaninetransferase (ALT), Gammaglutamiltranspeptidase (GGT), Alcaline Phosphatase, total Proteins and Albumin were determined. The extraction of plasma lipids was performed from lml. plasma, with 20 ml of ChloroformMethanol 2:1, according to Folch’s method (6). The brain was homogenised (Omni-Mixer. Omni.Waterbury, CT. USA) and a 1+0,2 grames aliquot was taken for lipid extraction according to gram. Folch’s method, with 20 ml of Chloroform-Methanol 2: 1 per each homogenised Brain lipid fractions were determined from the organ lipid extract. Polar lipids and neutral lipids were separated using cartridges of Sep-PakO Silica (Waters Chromat. Div. Millipore Co, MA. USA)(7). The dry extract corresponding the Polar Lipids was rediluted in chloroform (5 pg of extract per ~1) and 20 yl were deposited on 60A silica-gel plates, 20X20 (Whatman Int. Ltd. Maldstone, England). For a mobile phase and for 60 minutes a mixture of Chloroform-Methanol-Water-Acetic acid (60:30:6: 1) was used. The plate was revealed by using Molybdic Acid at 10% and it was heated on a stove at 12O’C for 30 minutes. The stains were semiquantified by means of a laser densitometry which was carried out using a plate reader and integrator SHIMADZU DR-13. CS-9OC!O (Shimatdzu. Kyoto, Japan). Palmitoilcerebroside (PC), Phosphatidilcholine (PhC), Phosphatidilserine (PhS) and Phosphatidiletanolamine (PhE) were determined. Plasmatic
FAs were determined
from the fatty extract which was taken from lml. of plasma; an inter-
BRAIN AND INTRALIPID OVERDOSE
883
nal standard, 1 ml of heptadecanoic acid (C-17) in hexane at a concentration of 0,6 mg/ml was added to. The whole solution was methylated by the addition of a boron trichloride in methanol at 10 % solution (Sigma-AIdrich.St. Louis, USA) and heating at 1OO’C for 30 minutes (8). The methylic esters were extracted by using hexane and they were analysed in a gas chromatograph Carlo Erba GC6000 Vega Series, with an ICU 600 processor (Carlo Erba Instruments. Milan, Italy) and equipped with a column packed with diehylenglycol on Cromosorb, 3% SP-23 10+2% SP-2300 and a flame ionization detector. The chromatographic conditions were the following ones: Injector temperature 150°C. Detector temperature, 17O“C. The temperature of the oven was programmed with an initial isotherm of 150°C for 3 minutes, followed by a 3”C/minute gradient until 220°C were reached and remaining for 1 minute to go on with another l”C/minute gradient up to 23O’C to remain at that point for 5 minutes. Miristic, palmitic, heptadecanoic, stearic, oleic, linoleic, linolenic and arachidonic acids were identified by comparison of their relative retention time with those of commercial standards (SigmaAldrich. St. Louis. USA). The methylation and the chromatographic determination of brain FFAA were perfommed by using the same methods and conditions already used for plasma, from an aliquot of the organ lipid extract. The sections of the liver tissue and peritoneal interpretation.
samples
were stained with hematoxiline-eosine
for its
Each and every group was compared using a non parametric test (one way MANOVA ) Multiple corn parisions were carried out, all of them based upon Kruskal-Wallis’rank sums.The difference between the groups was estimated by Dunn approximation (9)( 10). The confidence interval was 95 and 99%. RESULTS All 33 animals survived. Their average weigh increased during the experimental period (Table 1). When examining the abdominal cavity after 24 hours from the last infusion, only a milky aspect of the peritoneum was seen in the animals of IP group. The number, weigth and consistency of feces in all the animals including the IG group didn’t suffer changes all through the study. The plasma levels of glucose, urea and creatinine, were measured every 48 hours and they were normal levels. TABLE Animals
Weight (In grames). Group
lntraperitoneal group (n=9) Intravenous group (n=@ Intragastric group (n=8) Control Group (n=8)
1
Initial Weight 2.945 2.874 2.716 2.870
f + + +
Final Weight
262 109 166 70
2.974 2.908 2.876 3.120
& 378 t 178 & 130 + 91
Values are means f SD. The results after 15 days of infusion were: Total proteins, albumin, LDH and liver biochemistry mals. TABLE Liver
Biochemistry
2
(day 15th). IP (n=Y)
AST (U/I) ALT (U/l) GGT (U/I) T Bil.(mg/dl) D Bil (mg/dl) Ale Pho (mg/dl)
(Table 2) in plasma were normal in all the ani-
34+30 20 * 9 6&3 0,16 + 0,Ol 0,07 + 0,08 190 f 30
GROUPS IV (rl=B) 26 + 6 21 +6 4+2 0,12 -c 0,l 0,03 + 0,Ol 197,85
AST: Aspartatetransferase. ALT: Alaninetransferase. IP: Intraperitoneal, IV: Intravenous,. IG: Intragastric
IG (n=8)
CG (n=8)
13 *5 IS+2 5+1 0,14 + 0,08 0,06 It 0,02 211 k4.0
29 + 8 15*9 4tl 0,15 r 0,02 0,07 * 0,03 219k.5
GGT: Gammaglutamiltranspeptidase.Groups: and CG:Control. Values are means f SD.
E.M. BOTEJARA et al.
a84
The triglyceridemia reached higher values in those groups supplemented via parenteral route with significant differences respecting to IG and Control group. IP and IV groups were noticed to have suffered a significant elevation of the plasmatic phospholipids. The plasma values for total cholesterol also showed significant increases in the IP and IV groups respecting to the Control group. (Table 3).
TABLE 3 Plasmatics
Lipids. GROUPS IP
IV (n=8)
(n=9)
138+50 104*4O 167+50
Total Cholesterol (mg/dl) .. 107 + 25 ? Trigliceride (mgldl) . . . . . . . . . 325 + 55 7 Phospholipids (mgldl) ,.... 179 + 60 t
IG (n=8) t
CC (n=8)
65.1 f 5 68+7 107+6
7
43.7 + 9 62+ 14 80 + 6
IP: Intraperitoneal group. IV: Intravenous group. IG: Intragastric group. Values Statistics signification vs Control Group (CG): * (p
are means
f SD.
Palmitic, steak, oleic and linoleic acids in plasma rose in a significant way in the IP and IV groups. There were no significant rises in IG group. Both the increases in these FA and those in TG and PL, which were higher in IP group, were proportional to the lipids administered with the ITL (Table 4).
TABLE Plasma
Fatty
G
IP mp/ml El;; C-18 C-18:1 C-18:2 C-18:3 C-20:4
P/S:
(n=9) %
0.06 +O.Ol t 0.44 +0.03 7 0.2 *0.08 * 0.25 io.1 I 0.5 kO.01 * 0.02 ztO.01 0.08 *0.04
18118:l 18:2120:4 18:2/18:3
4
Acids
(4) (28) (13) (17) (32) (1) (5)
1.06 + 1.5 * 7 +2t 24 zt 6 * 1.1
IV ma/ml 0.04 0.2 0.1 0.15 0.36 0.1 0.02
(n=8) (%)
kO.01 kO.06 : kO.03 ? kO.03 +O.l * *0.03 Yto.02
(4) (21) (10) (16) (37) (10) (2)
0.7 f 0.1 i 4.6 + 2 18.3 f 6 1.4
ROUPS IG mgiml 0.02 0.13 0.05 0.09 0.15 0.01 0.07
(n=S) (%)
-co.01 i *0.18 t *0.09 t Lko.02 kO.05 kO.002 *O.Ol
(4) (25) (10) (17) (29) (2) (13)
0.5 zk 0.1 3.1 f 2.2 11.8 + 11 1.1
CG ma/ml 0.04 0.13 0.04 0.11 0.15 0.02 0.13
(n=8) (%)
kO.01 +0.03 *O.Ol *0.03 +0.05 *to.02 *O.Ol
(7) (21) (7) (18) (24) (3) (21)
0.06 21 9.5 29 32.5 4.15 0.1
0.3 f 0.1 1.4 f 1.2 8.2 f 10 1.5
IP: Intraperitoneal group. IV: Intravenous group. IG: Intragastric group. P/S: PolyunsaturedSatured FAs ratio.Values are means f SD . Percentages are based only in aritmetical means. Statistics signification vs Control Group (CC): * (p
The analysis of the lipid fractions from the brain (TABLE 5) showed descent PC values in IP and IV groups. The PhE is minor in the groups supplemented through parenteral route (IP and IV), in the same way that both PhC and PhS, down to the half of the CG levels. There was a decrease in the total of the lipid phosphatid fractions as a result of that. However, the proportion between polar and neutral lipids in brain all the four groups were very similar without any significant difference among them.
BRAIN AND INTRALIPID OVERDOSE
885
Table 5 Brain Lipids Fractions. GROUPS
PE ... . .. .. .. PhC+PhS . . PhE . . . . . . . . ... . .. PC PLINL . . . .
IP (n=9) r*n/IOO I*$J
IV (n=8) _MO/100~0
4-c3 13 f 10 7+5* 32 k 14 2&l
3+ 8+ 9+ 21 + I.5 f
3 5 9* 1s 0.6
IG (n=8) I*0/1001(0
CC (II=81 ug/10011e
IO+5 28 r10 27 +I4 39k 18 1.3 f 0.3
.5*2 21 +6 18+8. 37* II 1.7 + 0.6
PhE: Phosphatidiletanolamine. PhC: Phosphatidilcholine. PhS: Phosphatidilserine. PC: Palmitoilcerebroside. PUNL: Neutral lipids/Polar lipids ratio. IP: Intraperitoneal group. IV: Intravenous group. IG: lntragastric group Values are means f SD .Statistics signification vs Control Group (CG): * (~0.05)
When analysing the content of brain FAs (TABLE 6), we noticed small quantities of miristic acid undetectable in the CG. Palmitic acid (C-16) was noticed in the same proportion in all the groups, with smaller quantities in the intraperitonel group. Stearic acid (C- 18) and oleic acid (C-18: 1) showed substantially higher values in the three groups receiving the lipid supplement, with significant differences respecting to the CG.
TABLE Brain
Fatty
IP mglg c-14 C-16 C-18 C-18:1 C-18:2 C-20:4 18118: I 18:2/20:4 P/S
6
Acids.
(n=9) (%)
1.5 f 0.6 52 +14* _% t 10 * 65 k 16. 9 * 3 f 49 +I4
(1) (22) (24) (28) (4) (21)
0.9 + 0.1 t 0.2 t- 0.08 f. 0.5
IP: Intraperitoneal FAs ratio.Values cation vs Control
IV me/g
GROUPS (n=X) (%)
IG m g/g.
3.4 f 4 6Ok21 66+ 8. t 75 + 24 12+3 t 572 18
(I) (22) (24) (27) (4) (21)
I.1 + 0.2 102 + 44 * 66+21 * 152* 81_ 1S f 0.8 t 74 + 25
0.9 * 0. I t 0.2 f 0.1 i 0.5
group, IV: Intravenous Group (CG): * (~~0.05)
(0.2) (26) (16) (37) (4) (18)
CG
(n=n)
C% )
me/g
96 f 33 5+2 _s454+19 154+82 76 f 58
0.4 f 0.2* 0.2 f 0.02 -F
group. IG: Intragastric
are means f SD . Percentages
(n=8) (%)
(25) (1) (14) (40) (20)
0.1 + 0.07 2.2 + 0.9 2.2
1.3
group. P/S: Polyunsatured-Satured
are based only in aritmetical
means. Statistics
signifi-
and t (~0.01).
The amounts of linoleic acid (C- 18:2) in the supplemented groups, basically in parenteral route ones, were clearly lower to the CG. Arachidonic acid (C-20:4) was found in similar proportions in all groups, though quantities were smaller in IV and above all in IP groups. Linolenic acid was not detected. The ratio stearic/oleic was substantially greater in the three supplemented noleic/arachidonic decreased to significant levels in the four groups.
groups, whereas
the ratio li-
The microscopic study of the liver showed a marked and diffused microvesicular infiltration in the IV group animals. In the IP group only local alterations were observed (lipidic and mononuclear infiltration of the peritoneum and ingurgitation of lymphatics in the diaphagms, mesentery and intestinal wall).
886
E.M. BOTEJARA et
al.
DISCUSSION The plasmatic levels of lipids, after the infusion of IntralipidB 20%, follow a gradient of lineal or exponential plasmatic clearance, according to the overload they’ve received, as it was defined by Halberg (1 I). As the absortion through the peritoneal membrane happens gradually and fat still remains in the peritoneal cavity for several hours after the infusion, the increase of the plasmatic levels of TG and phospholipids are higher when intravenously administered. After the intravenous administration of chylomicrons there is a higher concentration during the first hours and, as a consequence, a greater plasmatic clearance. In fact, the trygliceridemia levels in the IV group are closer to the values in the IG and CG groups, rather than the levels of IP group. Another works have described similar findings (12)( 13). Hipercholesterolemia is attributed to the transfer of cholesterol and TG between the lipidic particles administered and the subject’s own particles.( 14). This is provoked by the lecitins of the administered emulsion which form stables micelles, if they are not quickly cleared. The higher amounts of phospholipids in our animals plasma in the groups IP and IV may contribute or rather, may be its own origin. However, this effect can’t only be attributted to the quantity of administered lipids. We have found rats and dogs in our laboratory with similar values, after injecting them with lower doses of lipids for 30 days (12)( 15). All these facts suggest to us a relationship with the lasting of the treatment, besides the effect dose-dependent, being this reported in human beings (16). There’s an elevation of the injected fatty acids with the ITL, which is higher in absolute terms in those groups receiving supplementation of lipids through the parenteral route and this suggests to us a realtionship with the administration route. The desaturation ratio C-l EXl8: 1 is similar in the three supplemented groups (IP, IV and IG), whereas the ratio C-18:2/C-20:4 decreases in this groups due to the decrease of the arachidonic acid. All this make us think that the administration of ITL has changed the distribution of plasmatic FAs in these animals, varying the proportions much farther than the ones observed in another similar experiments in rats that we have performed in our lab (13). In the comparision of the different fatty acids, there’s a greater amount in the supplemented groups, with a signit? cant difference between the parenteral groups and the CG and IG groups. The composition of the lipidic fractions and the fatty acids found in the IG group shows a more phisiological behavior of this route when lipidic overload. Since the plasmatic fatty acids values are a result of the hydrolisis of both ITL and the lipids in the tissues, we can’t evaluate to a what extent obeys the distribution at the moment of taking the sample. However, if we take in account the elongation-desaturation ratio, we could suppose that a part of the changes in the plasmatic distribution of FAs is due to the methabolism of these in the tissues. The relation PWNL in brain is similar in all the groups, though there’s a predominance of the polar lipids which they seem not to be affected by the overdosage of ITL. All the fractions (coline, serine and etalomine) of the polar lipids we have determined, and apported by ITL, decrease in a very important and significant way in the groups parenterally supplemented, compared to the CG and IG groups. However, another authors have found out that, after the administration of TPN with ITL. there’s a substantial increase of the etalomine and coline fractions in the brain of new born human beings (5). We believe that the reason for these divergency is the age of the different subjects. In different species, it has been demonstrated that the lipidic methabolism, not only oxidative but also structural of the brain is enormously “opened” and influenceable during the gestational period and during the different stages of growth. In fact, the neurological affectation, both basic and intellectual, is clearly affected by the lipids of the diet in the period of brain development (3)(4)( l7), which is radically different to what happen with an already formed brain, though some work suggests that the FAs composition of the adult brain its labile with respect to qualitative differences in dietary fat (18). We have tried to find out if the oversaturation with the lipids normally used in TPN produces changes in the composition of the already developped brain. In this sense, after the plasmatic overload by using different routes of infusion we have seen that the lipidic fractions of the brain don’t increase but rather decrease. At the same time, there’s an increase of the stearic and oleic acids in the parenterally supplemented groups, due to the overdosage of them. It doesn’t seem, however, that these FAs are incorporated structurally by the central nervous system cells, if we consider the decrease of lipidic fractions. On the contrary, there’s a marked decrease of C- 18:2 in all the supplemented groups and C-20:4 in the parenterally supplemented groups, despite of existing an excessive supply of linoleic, which is in great amounts in the ITL. This decrease seems to be produced by a derivation to oxidative phenomena, above all if we consider that it also decrease in a very inmportant way, its elongation product C-20:4. Anothers investigators have found important amounts of linoleic acid after the administration
BRAIN AND INTRALIPID OVERDOSE
887
of ITL in TNP in human new born brains (19)(20). The fact that we , after overdosage, don’t find it reaffirm US in the hypothesis suggested previously respecting to the influence of the madurative moment. The administered linoleic acid doesn’t incorporate to the cellular membranes in the already developed brain and as a result doesn’t induce changes in the brain lipidic fractions. This oxidative derivation has been demonstrated for the linolenic acid in several works with animals and in diverse circunstamces(21)( 17). According with our results, it is also possible that the excess of linoleic acid injected to the IP, IV and IG groups cause a greater synthesis of derivated from the ciclo oxygenase and peroxidase via (prostanoids, tromboxanes, leucotriens). In general, it seems clear that changes originated in the lipidic composition of the brain in the case of our adult animals are different and lower than those described for another animals and human lactants. Increases of structural lipids in the brain already developed are not produced after overload. On the other hand, though intraperitoneal absortion of ITL is produced through the subdiaphragmatic and mesenteric lymphatics and also have a clearance rhythm which is different to the ones of IV route, the results in both groups are similar. Only the enteral route is able to absorb the fat overload. In any case, it is necessary to develop when after overload through different mals.
studies to determine the final destination of brain lipids exactly routes, in different madurative moments and in different mam-
ACKNOWLEDGMENTS We thank J. Remon Alvarez-Arenas graphie.
and Victor Ruiz Garcia for your help in the thin layer chromate
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of linoleic
2 1) Cunnane SC, Chen ZY, Yang J, Liede AC, Hamadeh M, Crawford MA: a-Linolenic mans: A direct funcional role or dietary precursor?. Nutrition 1991 7437-39.
Accepted
for publication January 12, 1995.
acid
acid in hu-
Pergamon
EFFECT
OF INTRALIPIDB OVERDOSE ON BRAIN LIPID COMPOSITION IN THE ADULT RABBIT AND ITS RELATION WITH THE ROUTE OF ADMINISTRATION
MD, PhD’ ; JM Moran Pence, MD, PhDI; J. Salas Martinez, MD, PhD’; G. Mahedero Ruiz, MD, PhDl; G. Cruz Villal6n, ChDI; V Climent Mata, MD, PhD*; M. Molina FernAndez’, PhD. ‘Dep. Surgery (chairman: Prof LM Vinagre), *Dep Morphologic Sciences and 13Dep of Bioestatistics. Universidad de Extremadura, Badajoz. Spain. E. Macia Botejara,
ABSTRACT A group of 33 New Zeland rabbits were injected with 4g/kg weight/day of Intralipid (ITL) 20%. They were injected intravenously, intraperitoneally and intragrasticaily. The aim was to study the variations in plasma and brain lipids after fats overdosages. A control group, which was operated without receiving ITL, was also established. After IS days of daily lntralipid dosage, complex lipids and fatty acids (FA) administered with ITL rose in plasma levels in the intravenous and intraperitoneal groups. The study of brain lipids showed decreasing levels of phosphatidilethanolamine (PhE), phosphatidilcholine and phosphatidilserine (PhC+PhS), and palmitoil cerebroside (PC) in those groups parenterally supplemented with ITL, without any variation in the polar lipds/neutral lipids ratio (PUNL). Those groups supplemented with ITL showed increasing levels of the stearic (C-18) and oleic (C-18:1) acids in brain, whereas linoleic (C-18:2) and arachinodic (C-20:4) acids levels decreased. In conclusion, parenteral lntralipid overdosages cause longer -lasting changes in plasma lipids after intraperitoneal administration. A decreasing level of structural lipids, linoleic and arachidonic acids and also the ratio between both is noticed in the brain affecting all supplemented groups. All these changes in the adult animals are different to the ones described in other animals and lactant humans. Key words: Brain lipids; Lipids clearance;
lntraperitoneal
absorption;
Lipid overdose
INTRODUCTION Lipids are the essential constituents of the brain.They may become up to a 60% of the organ weight in dry conditions and they are mainly part of the membranous structures. Polyunsaturated fatty acids (PIJFAs) stand out among the brain fatty acids. They derivate from the linoleic (C-18:2) and the linolenic (C-183) acids. Their elongation and desaturation is mediated by the same enzyme systems and a competition between both is possible. This circumstance allows the brain lipids composition, and as a consequence, the neurological functions and the learning capacity, to be influenced by external contributions of fat, as it has been demonstrated in different studies (l)(2)(3)(4). Changes in brain lipid composition in lactant and premature babies, have been also demonstrated, when following the administration of total parenteral nutrition (TPN) with Intralipid (ITL), rich in lecithins and palmitic (C- 16), oleic (C- 18: 1). linoleic (C- l8:2) and arachidonic (C-20:4) acids (5). However. there are few works which, when studiying the possible changes in lipid body composition after TPN, differentiate the efects that they are due to the composition of the nutritional formula. to the access route of the Iipids(oral. parenteral), whether they are produced with dosage-dependent relation- after an overload and whether these possible changes once the brain is developed. Our aim in this work was to study the lipid composition of the general plasma pool and the brain after overdosage with ITL using different vias (enteral, intraperitoneal and intravenous) in a group of adult mammals.
’ Adress correspondence to: Enrique Macia Botejara. Department of Surgery. Facultad de Medicina. UEX. Av de Elvas s/n. 06071 BADAJOZ. Spain. This work was support for a grant from the Fondo de lnvestigaciones Sanitarias de la Seguridad Social (FISS) no 89/0867 and 91/0147. 881
E.M. BOTEJARA et al.
882
METHODS
AND
MATERIALS
Thirty-three New Zeland rabbits aged 6+1 months housed under the following conditions were used: 14-10 h. cycles of light/darkness, temperature from 20+ to 2” C and 13 renewals of air per hour. All through the experimental period, the animals received water and chow “ad libitum”. The composition of the diet was as follows: Proteins, 15%; Fat, 2,3%; Cellulose 17%; Ash 12%; Starch, 14% and Minerals 2,45%. The whole group was anaesthetised with Ketamina (KetolairO.Lab.Parke-Davis.Morris-Plains, N.J. USA), at doses of 25 mg/kg and preanaesthesia with Fenotiacina (CombelenB.Lab. Bayer, Germany) at doses of 1 ml/Kg. both intramuscularly administered. Experimental groups: Intraneritoneal Group (IP). This group was formed with 9 animals which underwent a minilaparotomy in the right hypocondrium to set a silastic catheter (NutricathB No. 18. Lab. Vigon) in the abde mini1 cavity which’reached the interscapular region and through which ITL at 26% was injected (Kabi Vitrum. Stockholm. Sweden)at a dose of 4 gr. of lipids/Kg/day, for 15 days and in two bolus (at 9.00 a.m. and 20.00 p.m.). Over ten centimeters of catheter were left inside the abdomen, and several drainage paths were opened in its five dystal centimeters. Intravenous Group (IV). This group was formed with 8 animals, to which a silastic catheter (NutricathO No 18. Lab. Vigon) was implanted in the external jugular vein and it was tunnelised to reach the interscapular region. The lipids administration was carried out under the same conditions and using the same doses alreadv described above for erouD IP. IntraeraGic Group GIG). This g&up was formed with 5 an:mals which received the lipids by means of a nasogastric tube Fr 5 made of noliurethane. The dailv amount of lipids received bv each animal was the same and the periods of time were also the same as the one ised in the other groups, but with a single bolus. A simulated surgical operation was practised before treatment started. Control Group (CC). This group was composed of 8 animals. They didn’t received ITL supplement, though they were operated under general anaesthesia to set an inserted catheter. All animals involved in the experiment were weighed at the beginning and at the end of the experiment apart from the clinical daily follow-up and to determine the glucose, urea and creatinine in blood every 48 hours. A 1Oc.c. extraction of blood from the central arteria of the ear was perfommed on the 16th day, 24 hours after the last infusion. Inmediately afterwards, the animals were anaesthetised and a medial laparotomy was carried out. A biobsy of the anterior edge of the major hepatic lobe, diaphragm and intestine was taken. After a medial craneotomy was performed, the encephalic mass and the cerebellum were extracted and maintained at -80°C until it was analysed. This analysis was to be done between the 48 and 72 following hours. Using a Hitachi 705 (Hitachi Ltd. Osaka, Japan) autoanalyser, the levels of Glucose, Urea, Total Cholesterol (TCh), Triglycerides (TG), Phospholipids (PL), total and direct Bilirubin, Aspartatetransferase (AST), Alaninetransferase (ALT), Gammaglutamiltranspeptidase (GGT), Alcaline Phosphatase, total Proteins and Albumin were determined. The extraction of plasma lipids was performed from lml. plasma, with 20 ml of ChloroformMethanol 2:1, according to Folch’s method (6). The brain was homogenised (Omni-Mixer. Omni.Waterbury, CT. USA) and a 1+0,2 grames aliquot was taken for lipid extraction according to gram. Folch’s method, with 20 ml of Chloroform-Methanol 2: 1 per each homogenised Brain lipid fractions were determined from the organ lipid extract. Polar lipids and neutral lipids were separated using cartridges of Sep-PakO Silica (Waters Chromat. Div. Millipore Co, MA. USA)(7). The dry extract corresponding the Polar Lipids was rediluted in chloroform (5 pg of extract per ~1) and 20 yl were deposited on 60A silica-gel plates, 20X20 (Whatman Int. Ltd. Maldstone, England). For a mobile phase and for 60 minutes a mixture of Chloroform-Methanol-Water-Acetic acid (60:30:6: 1) was used. The plate was revealed by using Molybdic Acid at 10% and it was heated on a stove at 12O’C for 30 minutes. The stains were semiquantified by means of a laser densitometry which was carried out using a plate reader and integrator SHIMADZU DR-13. CS-9OC!O (Shimatdzu. Kyoto, Japan). Palmitoilcerebroside (PC), Phosphatidilcholine (PhC), Phosphatidilserine (PhS) and Phosphatidiletanolamine (PhE) were determined. Plasmatic
FAs were determined
from the fatty extract which was taken from lml. of plasma; an inter-
BRAIN AND INTRALIPID OVERDOSE
883
nal standard, 1 ml of heptadecanoic acid (C-17) in hexane at a concentration of 0,6 mg/ml was added to. The whole solution was methylated by the addition of a boron trichloride in methanol at 10 % solution (Sigma-AIdrich.St. Louis, USA) and heating at 1OO’C for 30 minutes (8). The methylic esters were extracted by using hexane and they were analysed in a gas chromatograph Carlo Erba GC6000 Vega Series, with an ICU 600 processor (Carlo Erba Instruments. Milan, Italy) and equipped with a column packed with diehylenglycol on Cromosorb, 3% SP-23 10+2% SP-2300 and a flame ionization detector. The chromatographic conditions were the following ones: Injector temperature 150°C. Detector temperature, 17O“C. The temperature of the oven was programmed with an initial isotherm of 150°C for 3 minutes, followed by a 3”C/minute gradient until 220°C were reached and remaining for 1 minute to go on with another l”C/minute gradient up to 23O’C to remain at that point for 5 minutes. Miristic, palmitic, heptadecanoic, stearic, oleic, linoleic, linolenic and arachidonic acids were identified by comparison of their relative retention time with those of commercial standards (SigmaAldrich. St. Louis. USA). The methylation and the chromatographic determination of brain FFAA were perfommed by using the same methods and conditions already used for plasma, from an aliquot of the organ lipid extract. The sections of the liver tissue and peritoneal interpretation.
samples
were stained with hematoxiline-eosine
for its
Each and every group was compared using a non parametric test (one way MANOVA ) Multiple corn parisions were carried out, all of them based upon Kruskal-Wallis’rank sums.The difference between the groups was estimated by Dunn approximation (9)( 10). The confidence interval was 95 and 99%. RESULTS All 33 animals survived. Their average weigh increased during the experimental period (Table 1). When examining the abdominal cavity after 24 hours from the last infusion, only a milky aspect of the peritoneum was seen in the animals of IP group. The number, weigth and consistency of feces in all the animals including the IG group didn’t suffer changes all through the study. The plasma levels of glucose, urea and creatinine, were measured every 48 hours and they were normal levels. TABLE Animals
Weight (In grames). Group
lntraperitoneal group (n=9) Intravenous group (n=@ Intragastric group (n=8) Control Group (n=8)
1
Initial Weight 2.945 2.874 2.716 2.870
f + + +
Final Weight
262 109 166 70
2.974 2.908 2.876 3.120
& 378 t 178 & 130 + 91
Values are means f SD. The results after 15 days of infusion were: Total proteins, albumin, LDH and liver biochemistry mals. TABLE Liver
Biochemistry
2
(day 15th). IP (n=Y)
AST (U/I) ALT (U/l) GGT (U/I) T Bil.(mg/dl) D Bil (mg/dl) Ale Pho (mg/dl)
(Table 2) in plasma were normal in all the ani-
34+30 20 * 9 6&3 0,16 + 0,Ol 0,07 + 0,08 190 f 30
GROUPS IV (rl=B) 26 + 6 21 +6 4+2 0,12 -c 0,l 0,03 + 0,Ol 197,85
AST: Aspartatetransferase. ALT: Alaninetransferase. IP: Intraperitoneal, IV: Intravenous,. IG: Intragastric
IG (n=8)
CG (n=8)
13 *5 IS+2 5+1 0,14 + 0,08 0,06 It 0,02 211 k4.0
29 + 8 15*9 4tl 0,15 r 0,02 0,07 * 0,03 219k.5
GGT: Gammaglutamiltranspeptidase.Groups: and CG:Control. Values are means f SD.
E.M. BOTEJARA et al.
a84
The triglyceridemia reached higher values in those groups supplemented via parenteral route with significant differences respecting to IG and Control group. IP and IV groups were noticed to have suffered a significant elevation of the plasmatic phospholipids. The plasma values for total cholesterol also showed significant increases in the IP and IV groups respecting to the Control group. (Table 3).
TABLE 3 Plasmatics
Lipids. GROUPS IP
IV (n=8)
(n=9)
138+50 104*4O 167+50
Total Cholesterol (mg/dl) .. 107 + 25 ? Trigliceride (mgldl) . . . . . . . . . 325 + 55 7 Phospholipids (mgldl) ,.... 179 + 60 t
IG (n=8) t
CC (n=8)
65.1 f 5 68+7 107+6
7
43.7 + 9 62+ 14 80 + 6
IP: Intraperitoneal group. IV: Intravenous group. IG: Intragastric group. Values Statistics signification vs Control Group (CG): * (p
are means
f SD.
Palmitic, steak, oleic and linoleic acids in plasma rose in a significant way in the IP and IV groups. There were no significant rises in IG group. Both the increases in these FA and those in TG and PL, which were higher in IP group, were proportional to the lipids administered with the ITL (Table 4).
TABLE Plasma
Fatty
G
IP mp/ml El;; C-18 C-18:1 C-18:2 C-18:3 C-20:4
P/S:
(n=9) %
0.06 +O.Ol t 0.44 +0.03 7 0.2 *0.08 * 0.25 io.1 I 0.5 kO.01 * 0.02 ztO.01 0.08 *0.04
18118:l 18:2120:4 18:2/18:3
4
Acids
(4) (28) (13) (17) (32) (1) (5)
1.06 + 1.5 * 7 +2t 24 zt 6 * 1.1
IV ma/ml 0.04 0.2 0.1 0.15 0.36 0.1 0.02
(n=8) (%)
kO.01 kO.06 : kO.03 ? kO.03 +O.l * *0.03 Yto.02
(4) (21) (10) (16) (37) (10) (2)
0.7 f 0.1 i 4.6 + 2 18.3 f 6 1.4
ROUPS IG mgiml 0.02 0.13 0.05 0.09 0.15 0.01 0.07
(n=S) (%)
-co.01 i *0.18 t *0.09 t Lko.02 kO.05 kO.002 *O.Ol
(4) (25) (10) (17) (29) (2) (13)
0.5 zk 0.1 3.1 f 2.2 11.8 + 11 1.1
CG ma/ml 0.04 0.13 0.04 0.11 0.15 0.02 0.13
(n=8) (%)
kO.01 +0.03 *O.Ol *0.03 +0.05 *to.02 *O.Ol
(7) (21) (7) (18) (24) (3) (21)
0.06 21 9.5 29 32.5 4.15 0.1
0.3 f 0.1 1.4 f 1.2 8.2 f 10 1.5
IP: Intraperitoneal group. IV: Intravenous group. IG: Intragastric group. P/S: PolyunsaturedSatured FAs ratio.Values are means f SD . Percentages are based only in aritmetical means. Statistics signification vs Control Group (CC): * (p
The analysis of the lipid fractions from the brain (TABLE 5) showed descent PC values in IP and IV groups. The PhE is minor in the groups supplemented through parenteral route (IP and IV), in the same way that both PhC and PhS, down to the half of the CG levels. There was a decrease in the total of the lipid phosphatid fractions as a result of that. However, the proportion between polar and neutral lipids in brain all the four groups were very similar without any significant difference among them.
BRAIN AND INTRALIPID OVERDOSE
885
Table 5 Brain Lipids Fractions. GROUPS
PE ... . .. .. .. PhC+PhS . . PhE . . . . . . . . ... . .. PC PLINL . . . .
IP (n=9) r*n/IOO I*$J
IV (n=8) _MO/100~0
4-c3 13 f 10 7+5* 32 k 14 2&l
3+ 8+ 9+ 21 + I.5 f
3 5 9* 1s 0.6
IG (n=8) I*0/1001(0
CC (II=81 ug/10011e
IO+5 28 r10 27 +I4 39k 18 1.3 f 0.3
.5*2 21 +6 18+8. 37* II 1.7 + 0.6
PhE: Phosphatidiletanolamine. PhC: Phosphatidilcholine. PhS: Phosphatidilserine. PC: Palmitoilcerebroside. PUNL: Neutral lipids/Polar lipids ratio. IP: Intraperitoneal group. IV: Intravenous group. IG: lntragastric group Values are means f SD .Statistics signification vs Control Group (CG): * (~0.05)
When analysing the content of brain FAs (TABLE 6), we noticed small quantities of miristic acid undetectable in the CG. Palmitic acid (C-16) was noticed in the same proportion in all the groups, with smaller quantities in the intraperitonel group. Stearic acid (C- 18) and oleic acid (C-18: 1) showed substantially higher values in the three groups receiving the lipid supplement, with significant differences respecting to the CG.
TABLE Brain
Fatty
IP mglg c-14 C-16 C-18 C-18:1 C-18:2 C-20:4 18118: I 18:2/20:4 P/S
6
Acids.
(n=9) (%)
1.5 f 0.6 52 +14* _% t 10 * 65 k 16. 9 * 3 f 49 +I4
(1) (22) (24) (28) (4) (21)
0.9 + 0.1 t 0.2 t- 0.08 f. 0.5
IP: Intraperitoneal FAs ratio.Values cation vs Control
IV me/g
GROUPS (n=X) (%)
IG m g/g.
3.4 f 4 6Ok21 66+ 8. t 75 + 24 12+3 t 572 18
(I) (22) (24) (27) (4) (21)
I.1 + 0.2 102 + 44 * 66+21 * 152* 81_ 1S f 0.8 t 74 + 25
0.9 * 0. I t 0.2 f 0.1 i 0.5
group, IV: Intravenous Group (CG): * (~~0.05)
(0.2) (26) (16) (37) (4) (18)
CG
(n=n)
C% )
me/g
96 f 33 5+2 _s454+19 154+82 76 f 58
0.4 f 0.2* 0.2 f 0.02 -F
group. IG: Intragastric
are means f SD . Percentages
(n=8) (%)
(25) (1) (14) (40) (20)
0.1 + 0.07 2.2 + 0.9 2.2
1.3
group. P/S: Polyunsatured-Satured
are based only in aritmetical
means. Statistics
signifi-
and t (~0.01).
The amounts of linoleic acid (C- 18:2) in the supplemented groups, basically in parenteral route ones, were clearly lower to the CG. Arachidonic acid (C-20:4) was found in similar proportions in all groups, though quantities were smaller in IV and above all in IP groups. Linolenic acid was not detected. The ratio stearic/oleic was substantially greater in the three supplemented noleic/arachidonic decreased to significant levels in the four groups.
groups, whereas
the ratio li-
The microscopic study of the liver showed a marked and diffused microvesicular infiltration in the IV group animals. In the IP group only local alterations were observed (lipidic and mononuclear infiltration of the peritoneum and ingurgitation of lymphatics in the diaphagms, mesentery and intestinal wall).
886
E.M. BOTEJARA et
al.
DISCUSSION The plasmatic levels of lipids, after the infusion of IntralipidB 20%, follow a gradient of lineal or exponential plasmatic clearance, according to the overload they’ve received, as it was defined by Halberg (1 I). As the absortion through the peritoneal membrane happens gradually and fat still remains in the peritoneal cavity for several hours after the infusion, the increase of the plasmatic levels of TG and phospholipids are higher when intravenously administered. After the intravenous administration of chylomicrons there is a higher concentration during the first hours and, as a consequence, a greater plasmatic clearance. In fact, the trygliceridemia levels in the IV group are closer to the values in the IG and CG groups, rather than the levels of IP group. Another works have described similar findings (12)( 13). Hipercholesterolemia is attributed to the transfer of cholesterol and TG between the lipidic particles administered and the subject’s own particles.( 14). This is provoked by the lecitins of the administered emulsion which form stables micelles, if they are not quickly cleared. The higher amounts of phospholipids in our animals plasma in the groups IP and IV may contribute or rather, may be its own origin. However, this effect can’t only be attributted to the quantity of administered lipids. We have found rats and dogs in our laboratory with similar values, after injecting them with lower doses of lipids for 30 days (12)( 15). All these facts suggest to us a relationship with the lasting of the treatment, besides the effect dose-dependent, being this reported in human beings (16). There’s an elevation of the injected fatty acids with the ITL, which is higher in absolute terms in those groups receiving supplementation of lipids through the parenteral route and this suggests to us a realtionship with the administration route. The desaturation ratio C-l EXl8: 1 is similar in the three supplemented groups (IP, IV and IG), whereas the ratio C-18:2/C-20:4 decreases in this groups due to the decrease of the arachidonic acid. All this make us think that the administration of ITL has changed the distribution of plasmatic FAs in these animals, varying the proportions much farther than the ones observed in another similar experiments in rats that we have performed in our lab (13). In the comparision of the different fatty acids, there’s a greater amount in the supplemented groups, with a signit? cant difference between the parenteral groups and the CG and IG groups. The composition of the lipidic fractions and the fatty acids found in the IG group shows a more phisiological behavior of this route when lipidic overload. Since the plasmatic fatty acids values are a result of the hydrolisis of both ITL and the lipids in the tissues, we can’t evaluate to a what extent obeys the distribution at the moment of taking the sample. However, if we take in account the elongation-desaturation ratio, we could suppose that a part of the changes in the plasmatic distribution of FAs is due to the methabolism of these in the tissues. The relation PWNL in brain is similar in all the groups, though there’s a predominance of the polar lipids which they seem not to be affected by the overdosage of ITL. All the fractions (coline, serine and etalomine) of the polar lipids we have determined, and apported by ITL, decrease in a very important and significant way in the groups parenterally supplemented, compared to the CG and IG groups. However, another authors have found out that, after the administration of TPN with ITL. there’s a substantial increase of the etalomine and coline fractions in the brain of new born human beings (5). We believe that the reason for these divergency is the age of the different subjects. In different species, it has been demonstrated that the lipidic methabolism, not only oxidative but also structural of the brain is enormously “opened” and influenceable during the gestational period and during the different stages of growth. In fact, the neurological affectation, both basic and intellectual, is clearly affected by the lipids of the diet in the period of brain development (3)(4)( l7), which is radically different to what happen with an already formed brain, though some work suggests that the FAs composition of the adult brain its labile with respect to qualitative differences in dietary fat (18). We have tried to find out if the oversaturation with the lipids normally used in TPN produces changes in the composition of the already developped brain. In this sense, after the plasmatic overload by using different routes of infusion we have seen that the lipidic fractions of the brain don’t increase but rather decrease. At the same time, there’s an increase of the stearic and oleic acids in the parenterally supplemented groups, due to the overdosage of them. It doesn’t seem, however, that these FAs are incorporated structurally by the central nervous system cells, if we consider the decrease of lipidic fractions. On the contrary, there’s a marked decrease of C- 18:2 in all the supplemented groups and C-20:4 in the parenterally supplemented groups, despite of existing an excessive supply of linoleic, which is in great amounts in the ITL. This decrease seems to be produced by a derivation to oxidative phenomena, above all if we consider that it also decrease in a very inmportant way, its elongation product C-20:4. Anothers investigators have found important amounts of linoleic acid after the administration
BRAIN AND INTRALIPID OVERDOSE
887
of ITL in TNP in human new born brains (19)(20). The fact that we , after overdosage, don’t find it reaffirm US in the hypothesis suggested previously respecting to the influence of the madurative moment. The administered linoleic acid doesn’t incorporate to the cellular membranes in the already developed brain and as a result doesn’t induce changes in the brain lipidic fractions. This oxidative derivation has been demonstrated for the linolenic acid in several works with animals and in diverse circunstamces(21)( 17). According with our results, it is also possible that the excess of linoleic acid injected to the IP, IV and IG groups cause a greater synthesis of derivated from the ciclo oxygenase and peroxidase via (prostanoids, tromboxanes, leucotriens). In general, it seems clear that changes originated in the lipidic composition of the brain in the case of our adult animals are different and lower than those described for another animals and human lactants. Increases of structural lipids in the brain already developed are not produced after overload. On the other hand, though intraperitoneal absortion of ITL is produced through the subdiaphragmatic and mesenteric lymphatics and also have a clearance rhythm which is different to the ones of IV route, the results in both groups are similar. Only the enteral route is able to absorb the fat overload. In any case, it is necessary to develop when after overload through different mals.
studies to determine the final destination of brain lipids exactly routes, in different madurative moments and in different mam-
ACKNOWLEDGMENTS We thank J. Remon Alvarez-Arenas graphie.
and Victor Ruiz Garcia for your help in the thin layer chromate
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utilization
of linoleic
2 1) Cunnane SC, Chen ZY, Yang J, Liede AC, Hamadeh M, Crawford MA: a-Linolenic mans: A direct funcional role or dietary precursor?. Nutrition 1991 7437-39.
Accepted
for publication January 12, 1995.
acid
acid in hu-