- Email: [email protected]
Effects of polyunsaturated fatty acid diets on plasma lipids of patients with adrenomultineuronal degeneration, hepatosplenomegaly and fatty acid derangement
Jour/ud ~/ the Neurolot,,ica/Sciences, 1983, 62 : 67 76 Elsevier
67
EFFECTS OF P O L Y U N S A T U R A T E D F A T T Y ACID DIETS ON PLASMA LIPIDS OF P A T I E N T S W I T H A D R E N O M U L T I N E U R O N A L D E G E N E R A T I O N , H E P A T O S P L E N O M E G A L Y A N D FATTY ACID DERANGEMENT
JEFFREY K. YAO 1. KEVIN P. CANNON l, RALPH T. HOLMAN 3 and PETER JAMES DYCK I I Peripheral No'w" Center, Department ~ff'Neuro/o:o', :~lai'o Clinic and P~mndalion, Rochester. MN 55905 and 2The Horrrtel hTstilute, University o/ Minnesota, Austin, M N 55912 /U.S.A.) (Received 9 May, 1983) (Accepted 6 July, 1983)
SUMMARY
The effect on plasma fatty acid composition of 3 6 weeks feeding of standard diets supplemented with various co6 polyenoic fatty acids (18:2, 18:3. 2 0 : 3 or 2 0 : 4 ) was studied in two young brothers with multineuronal degeneration plus. These boys had mental retardation or maldevelopment, neurosensory hearing loss, retinitis pigmentosa, progressive muscular atrophy, hepatosplenomegaly and adrenal failure. The study objectives were to localize the site of metabolic block and to assess the safety and short-term clinical effect of dietary treatment. Our studies have shown that the low plasma levels of 20 : 4t06 can be corrected by feeding ethyl arachidonate and that no adverse effects were experienced. A diet enriched in ethyl linoleate produced no obvious increases of 18 : 2~:~6 metabolites, indicating that these patients do not have a linoleate deficiency in their ¢,J6 polyenoic fatty acid pathway. Lack of incorporation of 20 : &o6 and a retroconversion of 20:3~o6 to 18 : 2~6 after a dihomo-7-1inolenate-enriched diet suggest that a defect of A5 desaturase may be involved.
Key words: Adrenomultineuronal degeneration Fatty acid derangement Hepatosplenomegaly - Plasma lipids Polyunsaturated latO' acid diels
This work was supported in part by Center Grants from NINCDS (NS-14304) and MDA (MDA-12), by Mayo, Borchard, Upton and Gallagher Funds; by grant NS-18012 from N1NCDS; by Program Project Grant HL-08214, Program Projects Branch, Extramural Programs, National Heart, Lung and Blood Institute; and by the Hormel Foundation. A preliminary report of this work was presented at the 66th FASEB annual meeting, New Orleans, LA, April 18, 1982. Address reprint requests to: Jeffrey K. Yao, Ph.D. 0022-510X/83/S03.00 (t) I983 Elsevier Science Publishers B.V.
68 INTRODUCTION
We have reported on two male siblings with a previously unrecognized syndrome characterized by multineuronat degeneration, hepatosplenomegaly and adrenocorticat deficiency (Dyck et at. 1981). Biopsied tissues showed a consistent reduction of polyunsaturated fatty acids (PUFA), especially of 20 : 4a~6. but not of 18:2m6. Thus. the ratio of 20:4~6,18:2~o6. which provides a biochemical index of product substrate relationship in the o~6 polyenoic pathway, was significantly reduced in our patients. A decrease of serum linoleate with normal or slightly decreased levels o1" arachidonate, as encountered in patients with cystic fibrosis (Kuo et al. 1961: Kuo and Huang 1965: Lloyd-Still eI al. 1981). multiple sclerosis lBaker et al 1964: Love et al 1974) and hereditary neuropathy (Yao et al. 1976, 1978: Davigon et al. 1979), results in a normal or slightly increased ratio of 20 : ~o6/18 : 2eo6. This ratio is markedly increased in patients with essential fatty acid (EFA) deficiency due to the malabsorption of dietary 18 : 2co6 (Collins et al. 1971 : Holman 1970). The defect in the o~6 polyenoic pathway in these various neurologic disorders appears to be different from that of cases reported here. The purpose of the present study was to localize the site of metabolic block by feeding standard diets enriched with various o~6 polyunsaturated fatty acids and to assess the short-term safety and effect of such dietary manipulation with a view to longer duration trials. MATERIALS AND METHODS Case report The clinical features of the two affected brothers, aged 5 (III-7) and 8 (III-6) years, have recently been reported (Dyck et al. 1981). They failed t o thrive from infancy and have peripheral visual field loss due to retinitis plgmentosa, neurosensory hearing loss and mental maldevelopment or retardation. Additionally, they have progressive, symmetrical, distal lower limb muscular atrophy causing instability and an abnormality of gait. Both boys have hepatosplenomegaly and subclinical adrenal failure. Sural nerve biopsies revealed a normal density and diameter distribution of myelinated and unmyelinated fibers with mild ultrastructural abnormalities of myelinated fibers. Liver biopsies showed evidence of portal cirrhosis. Dietary trials' Both patients were placed on a standard diet, sometimes supplemented with various ~6 polyunsaturated fatty acids, on different occasions during a period of 1 year. The duration of each dietary trial was 3 weeks (or as indicated) because 10 ~ 14 daysare normally required for equilibration of plasma fatty acid composition (Spritz and Mishkel 1969). The composition of each diet was as follows: (1) Standard diet: A cholesterol restricted diet (Mayo Clinic" Diet Manual)
69 was used to provide baseline lipid conditions prior to other dietary studies. The older boy (age 8) received a diet consisting of 52 g protein, 224 g carbohydrate and 54 g t;at (30 35'/~,, lipid diet, 9 g saturated and 45 g polyunsaturated), total calories 1645. The younger boy (age 5) received 42 g protein, 188 g carbohydrate, 48 g fat (7 g saturated and 41 g polyunsaturated), total calories 1375 (as recommended by Dr. Wayne Callaway and Ms Huse, Department of Internal Medicine, Mayo Clinic). (2) Standard diet supplemented with 18. 2~o6: 2 g of 701~, ethyl linoleate (isolated from hog liver by NuChek, Elysian, MN) from freshly opened vials, sealed under nitrogen, was blended into food and drink and eaten directly after preparation to minimize fatty acid oxidation. (3) Standard diet supplemented with 18 : 3o~6 : 450 mg of evening primrose oil (EPO) containing 9')~ of 18 • 3co6 was supplemented. Since EPO is a seed oil, its use here was that of a food. (4) Standard diet supplemented with 20 . 3co6 : A naturally enriched source of 20 : 3~o6(dihomo-7-1inolenic acid or DHLA) was not available. Therefore, synthetic DHLA (Hoffman-LaRoche, UK) was substituted. A limited clinical trial of DHLA had been performed by Hoffman-LaRoche company (Stone et al. 1979) in the United Kingdom, but not in the U.S.A. Thus, special approval from the Food and Drug Administration, U,S.A., was obtained for this dietary trial (IND # 18079). The dosage of D H L A for each patient was as follows: 1 capsule (50 mg DHLA containing 0.1',}o butylated hydroxyanisole and 0.I"~i butylated hydroxytoluene as anti-oxidants) at breakfast daily for the first 2 weeks, 1 capsule at breakfast and supper daily for the following 2 weeks and 2 capsules at breakfast and supper and 1 capsule at lunch daily for the last 2 weeks. (5) Standard diet supplemented with 20 : 4co6" 2 g of 70~ ethyl arachidonate (isolated from hog liver by NuChek) was supplemented as in the standard diet with 18 : 2~o6. Lipid analysis The plasma (overnight fast) lipid and fatty acid compositions were analyzed on 2 or 3 consecutive days before and after each dietary trial. Lipid extraction from total plasma was performed according to the procedure of Nelson (1975). Nonpolar lipid subclasses and total phospholipids were separated from plasma lipid extracts by thin-layer chromatography (TLC) using hexane/diethyl ether/acetic acid (112: 35"2.5, v/v/v) as the developing solvent system (Yao et al. 1976). Following separation by TLC, a quantitative analysis of cholesteryl ester, triacylglycerol, free fatty acid, cholesterol and phospholipids was performed using a modified liquid scintillation counting procedure (Shand and Noble 1980). The TLC plate was first charred at 180°C for 15 min after spraying with 3% (w/v) cupric acetate in 8°o (w/v) phosphoric acid (Fewster et al. 1969). The charred bands were scraped off into scintillation vials and then suspended in 4 ml of distilled water. After the addition of 10 ml of Ready-Solv MP (Beckman, Irvine, CA 92713) scintillation cocktail, the vials were shaken vigorously to trap the charred lipids in the resultant
70 firm gel. The amount of quenching due to the presence of the charred lipids was measured by counting each sample for 5 rain in the external standard channels ratio (ESCR) mode of the liquid scintillation counter (Model LS-333. Beckman. Fullerton. CA 92634). The relationship between the ESCR and mass was linear up to at least 50/~g for all of the common lipid classes. Cholesteryl ester and triacylglycerol were transesterified using sodium methoxide reagent (Luddy et al. 1960~. Free fatty acid. phosphatidylcholine and total phospholipids were methylated with boron triftuoride/methanol reagent according to the method of Morrison and Smith (1964). The resulting fatty acid methyl esters were further purified by TLC. using petroleum ether/diethyl ether/acetic acid ( 9 0 : 1 0 : [ . v/v/vl as the developing solvent system (Metcalfet al. 1966). All methylated samples were analyzed on a Packard Gas Chromatograph model 419, equipped with dual hydrogen detectors. The operating conditions for gas-liquid chromatography (GLC) were as described previously (Yao et al. 1976~. Peaks on the chromatograms were identified by comparing the retention times with those of a variety of reference standard mixtures (Supelco. Inc.. Bellefonte, PA) and individual standards (Nu Check Prep. Elysian. MN and Analabs. Inc., North Haven. CT) and were calculated using the Autolab System IV computing integrator.
Follow-up evaluation The patients were medically assessed at weekly intervals throughout the study by (a) physical examination: (b) hemoglobin, leucocyte count and differential: (c) chemistry group; (d) complete urinalysis and (el chest X-ray. No attempt was made to ascertain clinical improvement because of the short treatment period. We were testing for biochemical restoranon only. RESULTS
The concentrations of the major plasma lipid subclasses after dietary trials are shown in Table 1. Among all the diets tested, only the standard diet-enriched in ethyl arachidonate substantially increased the plasma lipid concentration of both patients, particularly cholesterol and phospholipids. The percentage distribution of cholesteryl ester to total cholesterol remained essentially unchanged throughout the period of dietary trials. The ethyl arachidonate-enriched diet not only increased the arachidonate (20:40)6) content of plasma phosphatidylcholine for both patients (Fig. t), but also increased the proportion of 20:40)6 to total fatty acids 2.5-4.8-tbld in both cholesteryl ester and phospholipid fractions (Table 2). Concomitantly, there was a sharp decrease of 18 : 20)6 in both fractions. The proportion of 22 : ~06 in plasma phospholipids was slightly increased while that of 20 : 3o6 remained unchanged. The evening primrose oil-enriched diet produced no apparent increase of 18 : 30)6 in plasma phosphotipids (Table 3t. However. the ratio of20:3a,'6~18 : 3a,'6 was found to be substantially higher in both patients after the standard diet enriched with evenmg primrose oil compared with the standard diet alone.
FABLE 1 EI:EECT OF VARIOUS P O L Y U N S A T U R A T E D FATTY A C I D - E N R I C H E D DIETS ON PLASMA LIPID C O N C E N T R A T I O N S
Plasma lipids
Subjecl
Standard diet
Standard diet plus Ethyl linoleate
Phospholipid
111-7 111-6
Cholesterol
I11-7 II1-6
Triacylglycerol
Free fatty acid
85 _+ 3" 89 + 6
Evening primrose oil
Dihomo-> linolenate
Ethyl arachidonale
103 h 107
79 80
99 70
132 117
152 _+ 9 149 + 8
178 161
136 114
215 113
256 194
111-7 III-6
60 + 2 46 _+ 7
78 67
55 34
83 31
80 65
111-7 111-6
22 + 2 20 _+4
17 19
18 20
25 19
27 29
:' Mean value Img/dl) and SD of 3 samples obtained from patients on 3 different occasions after a standard diet. h Mean value of 2 samples obtained on 2 consecutive days after designated diet.
14
.
.
.
.
121-7
B
4
2
8 7 6 5 4 5 2 1 0
18:2 20:3 20"4 ~o~ P O L Y E N O I C FATTY ACIDS = Standard diet = Standard diet enriched in ethyl araehidonate = Standard diet enriched in d i h o m o - ~ q i n o l e n a t e
Fig. l. Effect of standard diet supplemented with either ethyl arachidonate or dihomo-7-1inolenatc (DHLA~ on 18 : 2uJ6, 20:30)6 and 20 : &o6 contents of plasma phosphatidylcholine. Phospholipid fatty acids were quantified by gas chromatography using methyl pentadecanoate as an internal standard. Each bar represents the mean of 2 or 3 samples obtained from patients on 2 or 3 consecutive days after a designated diet. 111-7 and 111-6 represent 2 affected patients.
72 After feeding both patients a DHLA-enriched diet. the proportion of 18 : 2co6 was elevated consistently in both free and esterified fatty acid fractions (Tabte 4J. This increase was accompanied by a decrease o f 18 : lco9. Furthermore. the ratio o f 20 : 4o96/20:3co6 in plasma lipids was suppressed to one half to one third of that control value (obtained after standard diet).
TABLE 2 E F F E C T OF L I N O L E A T E A N D A R A C H 1 D O N A T E - E N R t C H E D D IETS O N P L A S M A c~6 POLYENOIC FATTY ACIDS
Fatty acids
Standard diet
Cholesteryl
Standard diet plus Ethyl linoleatc
Ethyl a ra c hi dona t e
II1-"
1II-6
I[1-7
111-6
III-7
Il t -6
56.8 _* l . P 1 4.1 _ 0 . 1
56.5 z 1.4 4.4 z 0.2
61.1 ___ 1.7 4.1 ___0.4
58.9 -+ 2.2 4.3_+0.2
38.2 z 0.5 18.3 z 0.4
39.4 -+ 0.3 t5.1 z 0 . 3
24.5 ~- 0.2 3.2_+0.3 6.2-1-0.3 1.0 -+ 0.1
2 6 3 z 1.6 3.0_+0.1 7.5_*0.7 1.1 z 0 . 1
29.5 z 1.9 3.0 ~_ 0.4 6.6m 1.1 1.0 ~ 0.2
27.9 ~ 1.4 3.1 ___0.3 7.6-,-0.9 1.1 ~_0.1
14." m 1.6 2.8±0.1 20,6_+0.6 1.9 _T0.2
14.7 _+_1.4 2.9_+0.4 19.0-+1,] 1,8 ~_ 0.2
ester
18 : 2 20:4 Phospholipids
18 : 2 20:3 20:4 22:4
° Mean value (wt %) and SD of 3 samples obtained on 3 consecutive days. The saturated and m o n o u n s a t u r a t e d fatty a c i d s w e r e not included in tabulation.
TABLE 3 E F F E C T OF S T A N D A R D D I E T E N R I C H E D IN E V E N I N G P R I M R O S E O I L (EPO) O N (06 P O L Y E N O I C F A T T Y A C I D S OF P L A S M A P H O S P H O L I P I D S .
.
.
Fatty acids
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Standard diet -
18 : 2 18 : 3 20 : 3 20 : 4 20 : 3, 18 : 3
.
-
EPO
II1-7
11I-6
1II-7
111-6
24.62 a 0.35 2.89 5.54 8.26
25.46 0.38 2.24 6.76 5.89
24.36 0.29 3.56 6.04 12.28
25.82 0.32 3.05 7.27 9.53
Mean value (wt ~,(,) of 2 samples obtained on 2 consecutive days.
73 TABLE 4 EFFECT OF D I H O M O - 7 - L I N O L E N I C ACID (DHLA) DIET ON PLASMA FATTY ACID COMPOSITION
Fany acids ~'
Free fatty acids 111-7
16:0 18:0 18 : hJJ9 18 : 2oj6 20 : 3u06 20 : &o6 20 : 4eJ6,' 20 : 3c96
Cholesteryl ester 111-6
1II-7
Phospholipids 111-6
111-7
111-6
Std t,
DHLA
Std
DHLA
Std
DHLA
Std
DHLA
Std
DHLA
Std
DHLA
6.4" 12.8 49.5 23.4 0.6 0.8
6.2 13.2 34.2 35.5 1.3 1.6
16.1 11.8 45.9 19.3 0.5 0.5
19.1 7.9 28.2 33.7 0.5 0.6
7.8 1.4 21.0 62.1 0.5 3.8
8.9 0.8 15.3 68.7 1.0 3.1
6.1 0.7 17.8 67.7 0.5 4.4
6.2 0.7 12.4 75.2 0.9 3.3
27.4 14.1 16.8 30.6 2.2 4.3
27.1 14.3 9.8 34.3 4.7 4.6
21.6 14.6 15.2 ~5. ) 2.7 5.4
21.6 15.3 9.6 40.6 4.4 3.5
1.3
1.2
1.0
1.2
7.6
3,1
8.8
3.7
2.0
1.0
2.0
0.8
~' Minor fany acids, 14:0, 16: 1, 17:0, 18:3, 20:0, 20: 1, -~0 '~..~, 21:0, 22:4, ,,'~:5, ,_'~'~:6, 2 3 : 0 and 2 4 : 0 . were not tabulated. I~ Both patients (11I-7 and Ill-6) were placed on a standard (std) diet for 3 weeks just prior to the D H L A trial. L, Each value (wt <',,) represents the mean of 2 samples obtained on 2 consecutive days afier the designated diet.
DISCUSSION
These studies have shown that the low levels of plasma 20 : 4co6 encountered in our patients with multineuronal degeneration, hepatosplenomegaly and adrenal failure (Dyck et al. 1981) can be raised above normal using a diet enriched in ethyl arachidonate. No adverse clinical or laboratory findings were recognized, indicating that a long-duration trial of ethyl arachidonate may be possible. The marked increase of 20:&o6 was found at the expense of 18:2~o6 in both plasma cholesteryl ester and phospholipid fractions. A similar finding has been demonstrated in rat plasma after an ethyl arachidonate diet (Danon et al. 1975). This dramatic decrease in 18:2o)6 cannot be a simple dilution effect, but suggests that there is competitive incorporation between substrate and product in the ~o6 polyenoic fatty acid pathway. Arachidonate appears to be the preferred substrate for fatty acid esterification. Lack of accumulation of linoleate also suggests that there is no reverse conversion of arachidonate to linoleate (Mohrhauer and Holman 1963; Rahm and Holman 1964). We (Dyck et al. 1981) have previously shown that these patients have a normal plasma composition of 18 : 2~o6. Alter the standard diet was supplemented with ethyl linoleate, there was no apparent increase of any desaturated or elongated products further supporting the fact that these patients did not have a substrate deficiency in their cJ6 polyenoic fatty acid pathway to account for the decreased concentration of 20 : 4~o6. When 18 : 3~/J6 was fed to rats, no significant amounts of this acid could be detected in their livers (Rahm and Holman 1964; Sprecher 1974). The 18:3~o6
74 was rapidly elongated to 2 0 3~96 (Sprecher 1977). An enhanced, but insignificant. level of 18 • 3a~6 was found in the liver when 18 • 3co6 was fed to rats with essential fatty acid deficiency (Garcia and H o l m a n 1965). Therefore. lack of incorporation of 18 " 30,'6 into plasma lipids in these two patients, after a standard diet supplemented with evening primrose oil (Table 3), was probably due to a rapid elot~gation of 18 • 3o,'6 to 20 : 3co6. An increased ratio of 2 0 3co6J18 • 30,,6 (Table 3) supports this idea. Because the p r o p o r u o n of 20" 3~o6 generally is found to be much lower m mammalian tissue than that of 20 : 4v06. the principal biosynthetic role of 20 : 3~J6 is considered to be that of a precursor for 20:4eo6 (Sprecher 1977). Therefore. it ~s not surprising to find a low incorporation of 20 : 3~o6 in these two patients after a standard diet supplemented with D H L A Lack of increase of plasma 20 : 4e~6 by supplements of either ethyl linoleate or D H L A suggests that a defect of A5 desaturase may be involved. On the other hand, there was an increase of both free and esterified plasma linoteate (18"2eo6) content after the D H L A - e n r i c h e d diet (Fig. l and Table 4). indicating that a retroconversion of 20:3a~6 to 18:2co6 is active in these two patients. Whether this retroconversion is also present in healthy subjects is unknown. However. when either D H L A ester or D H L A was administered to rats or rabbits. the level of plasma linoleate of cholesteryl ester and phospholipids was significantly reduced (Danon et al. 1975 Oelz et al. 1976: Stone et al. 1979). Therefore. a retroconversion of20:3~o6 to 18 - 2c06 may be a key step in limiting A5 desaturasc activity in these two patients. In fact, various evidence (Verdino et al. 1964: Sprecher 1967. 1977) supports the hypothesis that termination ot" polyunsaturated fatty acid biosynthesis ~s regulated by a balance between chain elongation and desaturation reactions versus retroconversion. We have also reported a small, but unequivocally elevated amount of branchedchain fatty acids, phytanic and pristanic acids, in these two patients' plasma fYao et al. 1982). Their presence was not effected by dietary supplementation of various a~6 potyenoic fatty acids. REFERENCES Baker. R.W.. R. H. T h o m p s o n and K. J. Zilkha {19641 Serum f a n y acids in multiple scler~si,..I. Neurol. Neurosurg. Pa3'ehiat.. 27:408 414, Collins, F.D.. A.J. Sinclair. J.P. Royle, D . A , Coats, A . D Maynard and R . F . L c o n a r d (1971) Plasma lipids in h u m a n linoleic acid deficiency, Nutr. Metab., 13: 150--167. Danon. A.. M. Heinberg and J.A. Oates (1975] Enrichment of vat tissue lipids with fatty acids that are prostaglandin precursors, Bioehim. Biophys. Acta, 388:318 330, Davigon. J.. Y.S. Huang, J.P. Wolf and A Barbeau (1979) Fatty acid profile of major lipid classes m plasma lipoproteins of patients with Friedreich's ataxia Demonstration o f low tinoleic acid content most evident in the cholesterol-ester fraction. Can. J. Neurol, Sci., 6 : 2 7 5 293 Dyck. P.J.. J. K Yao. D . E Knickerbocker, R . T . Hotman, M . R . Gomez, A, B. Hayles and E.H. Lambert t198t) Multisystem neuronal degeneration, hepatosplenomegaly, and adrenoconical deficiency associated with reduced tissue arachidonic acid. Neurology (Minneap,}. 31 : 925 934. Fewster. M.E. B,J. Burns and J.F. Mead (1969] Q u a n m a t i v e densitometric thin-layer chromalography of lipids using copper acetate reagent. J. Chromatogr., 43: 120-126.
75 (iarcia, P . I . and R.T. Holman (1965) Competitive inhibitions in the metabolism of polyunsaturated fatty acids studied via the composition of phospholipids, triglycerides and didacteryl esters of rat tissues, J. Amer. Oil Chem. Sue., 42: [ 137 1141. Holman. R.T. (1970) Biological activities of and requirements for polyunsaturated acid. In: R.T. Holman (Ed.). l'ro~,,rcss m the Clwnfistrl o/ t.~IL~ and O/her Lipi&', l'o/. 9, Pergamon Press Ltd., Oxlord. pp. 606 682. Kuo. P.T. and N.N. Huang (1965) The tit"cot of medium chain triglyceride upon fat absorption and plasma lipid and deposition of fat in children with cystic fibrosis of the pancreas, J. ('li~t. hn'esl., 44:1924 1933. Kuo. P T . . N.N. Huang and D. R. Bassett [196l) the laity acid composition of the scram chylomicrons and adipose tissue of children wilh cystic fibrosis of the pancreas..I. Pcdkl/.. 60:394 403. I.Ioyd-Sli[l, ,1.1)., S. B..Iohnson and R.T. Holman (1981) Essential Iatty acid status in cystic fibrosis and the effects of safflower oil supplementation, Amer, J. ('lhl. Nulr., 34:1 7. Love, W.C., A. Cashell, M. Reynolds and N. Callaghan (1974) Linoleate and lhtty acid patterns of serum lipids m multiple sclerosis and other diseases, Brit. Med. J., 111:18 21. Luddy. F.E., R.A. Barford and R.W. Riemenschneider (1960) Direct conversion of lipid components to their larry acid methyl esters, J. Miner. Oil ('lwm., 37:447 451. Metcalf, L.D.. A.A. Schmitz and J.R. Polka (1966) Rapid preparation of fatty acid esters from lipids Ior gas chromatographic analysis, 4mtlrt. Chem., 38:514 515. Mohrhauer, H. and R.T. Holman (1963) Effects of dietary essential fatty acids upon polyunsaturated lhtty acids m rat heart tissue. In: A.C. Frajer (Ed.), Biochemistry Prohlems O( LipkL~', Elsevier Publishing Company. Amsterdam, pp. 446 452. Morrison, W.R. and L.M. Smith (1964) Preparation of fatty acid methyl esters and dimethyIacetals froln lipids with boron lluoride methanol. J. Lipid Re,s., 5:61)0 608. Nelson. G.J. (1975) Isolation and purification of lipids from animal tissues. In: E.G. Perkins (Ed.), .4mt@s'i.~ O~ Lipi& and Lipoproteh~s, American Oil Chemists" Society, Champaign, IL, pp. 1 22. Oelz, O.. H.W. Seyberth. H.R. Knapp, B.J. Sweetman and J.A. Oates (1976) Effects of Ik'eding cthyl-dihomo-;'-linolenatc on prostaglandin biosynthesis and platelet aggregation in the rabbit, Bi(whint. BIol)h.vs. .4eta, 431:268 277. Rahm. J.J. and R.T. Holman (1964) Studies of the metabolism of polyunsaturated acids by shortterm expcrimcnts. J. Nutr.,84:149 154. Shand, H. and R.('. Noble /1980) Quanlification of lipid mass by a liquid scintillation counter procedure I'ollouing charring on thin-layer plates, Anahv. Biochem.. 101: 427-434. Sprecher. H. (1967) ]"he total synthesis and metabolism of 7,10,13,16-docosatetracnoate in the rat, Biochinl. Biophy~. At/a, 144:296 304. Sprecher, H. (1977) Biosynthesis of polyunsaturated fatty acids and its regulation. In: W.-H. Kunau and R. T. Holman (Eds.), Polvunsaturawd l,'atly Act&, American Oil Chemists" Society, Champaign. 11., pp. l 18. Spritz, N. and M.A. M ishkel (1969) Effects of dietary Ihts on plasma lipids and lipoproteins An hypothesis fbr the lipid-lowering effect of unsaturated fatty acids, J. Clin. lnvesh. 48: 78-86. Stone, K.J.. A.L. Willis, M. Hart. J.J. Kirtland, P.B.A. Kcrnoff and G.P. McNicol (1979) ]"he metabolism ot'dihomo-i'-linolenic acid in man, Lipi&, 14: 174-180. Verdino, B., M.L. Blank. O.S. Privett and W.O. Lundberg (1964) Metabolism of 4,7.10,13,16docosapentaenoic acid in the essential fatty acid-deficient rat, J. Nutr.. 83:234 238. Yao. J.K. and P.J. Dyck (1978) Lipid abnormalities in hereditary neuropathy, Part 2 (Serum phospholipids), J. Nem'ol. Sci., 36:225 236. Yao, J. K., R. D. Elle£'~on and P.J. Dyck (1976) Lipid abnormalities in hereditary neuropathy. Part 1 iScrum non-polar lipids), J. Neuro/. Sci., 29:161 175. Yao. J.K., 1. Jardine and P.J. Dyck (1982) Presence of plasma branched-chain l'atty acids in multineuronal degeneration, hepatosplenomcguly and adrcnocortical insufficiency, ,I. Neurol. Sci., 55: 185 195.
67
EFFECTS OF P O L Y U N S A T U R A T E D F A T T Y ACID DIETS ON PLASMA LIPIDS OF P A T I E N T S W I T H A D R E N O M U L T I N E U R O N A L D E G E N E R A T I O N , H E P A T O S P L E N O M E G A L Y A N D FATTY ACID DERANGEMENT
JEFFREY K. YAO 1. KEVIN P. CANNON l, RALPH T. HOLMAN 3 and PETER JAMES DYCK I I Peripheral No'w" Center, Department ~ff'Neuro/o:o', :~lai'o Clinic and P~mndalion, Rochester. MN 55905 and 2The Horrrtel hTstilute, University o/ Minnesota, Austin, M N 55912 /U.S.A.) (Received 9 May, 1983) (Accepted 6 July, 1983)
SUMMARY
The effect on plasma fatty acid composition of 3 6 weeks feeding of standard diets supplemented with various co6 polyenoic fatty acids (18:2, 18:3. 2 0 : 3 or 2 0 : 4 ) was studied in two young brothers with multineuronal degeneration plus. These boys had mental retardation or maldevelopment, neurosensory hearing loss, retinitis pigmentosa, progressive muscular atrophy, hepatosplenomegaly and adrenal failure. The study objectives were to localize the site of metabolic block and to assess the safety and short-term clinical effect of dietary treatment. Our studies have shown that the low plasma levels of 20 : 4t06 can be corrected by feeding ethyl arachidonate and that no adverse effects were experienced. A diet enriched in ethyl linoleate produced no obvious increases of 18 : 2~:~6 metabolites, indicating that these patients do not have a linoleate deficiency in their ¢,J6 polyenoic fatty acid pathway. Lack of incorporation of 20 : &o6 and a retroconversion of 20:3~o6 to 18 : 2~6 after a dihomo-7-1inolenate-enriched diet suggest that a defect of A5 desaturase may be involved.
Key words: Adrenomultineuronal degeneration Fatty acid derangement Hepatosplenomegaly - Plasma lipids Polyunsaturated latO' acid diels
This work was supported in part by Center Grants from NINCDS (NS-14304) and MDA (MDA-12), by Mayo, Borchard, Upton and Gallagher Funds; by grant NS-18012 from N1NCDS; by Program Project Grant HL-08214, Program Projects Branch, Extramural Programs, National Heart, Lung and Blood Institute; and by the Hormel Foundation. A preliminary report of this work was presented at the 66th FASEB annual meeting, New Orleans, LA, April 18, 1982. Address reprint requests to: Jeffrey K. Yao, Ph.D. 0022-510X/83/S03.00 (t) I983 Elsevier Science Publishers B.V.
68 INTRODUCTION
We have reported on two male siblings with a previously unrecognized syndrome characterized by multineuronat degeneration, hepatosplenomegaly and adrenocorticat deficiency (Dyck et at. 1981). Biopsied tissues showed a consistent reduction of polyunsaturated fatty acids (PUFA), especially of 20 : 4a~6. but not of 18:2m6. Thus. the ratio of 20:4~6,18:2~o6. which provides a biochemical index of product substrate relationship in the o~6 polyenoic pathway, was significantly reduced in our patients. A decrease of serum linoleate with normal or slightly decreased levels o1" arachidonate, as encountered in patients with cystic fibrosis (Kuo et al. 1961: Kuo and Huang 1965: Lloyd-Still eI al. 1981). multiple sclerosis lBaker et al 1964: Love et al 1974) and hereditary neuropathy (Yao et al. 1976, 1978: Davigon et al. 1979), results in a normal or slightly increased ratio of 20 : ~o6/18 : 2eo6. This ratio is markedly increased in patients with essential fatty acid (EFA) deficiency due to the malabsorption of dietary 18 : 2co6 (Collins et al. 1971 : Holman 1970). The defect in the o~6 polyenoic pathway in these various neurologic disorders appears to be different from that of cases reported here. The purpose of the present study was to localize the site of metabolic block by feeding standard diets enriched with various o~6 polyunsaturated fatty acids and to assess the short-term safety and effect of such dietary manipulation with a view to longer duration trials. MATERIALS AND METHODS Case report The clinical features of the two affected brothers, aged 5 (III-7) and 8 (III-6) years, have recently been reported (Dyck et al. 1981). They failed t o thrive from infancy and have peripheral visual field loss due to retinitis plgmentosa, neurosensory hearing loss and mental maldevelopment or retardation. Additionally, they have progressive, symmetrical, distal lower limb muscular atrophy causing instability and an abnormality of gait. Both boys have hepatosplenomegaly and subclinical adrenal failure. Sural nerve biopsies revealed a normal density and diameter distribution of myelinated and unmyelinated fibers with mild ultrastructural abnormalities of myelinated fibers. Liver biopsies showed evidence of portal cirrhosis. Dietary trials' Both patients were placed on a standard diet, sometimes supplemented with various ~6 polyunsaturated fatty acids, on different occasions during a period of 1 year. The duration of each dietary trial was 3 weeks (or as indicated) because 10 ~ 14 daysare normally required for equilibration of plasma fatty acid composition (Spritz and Mishkel 1969). The composition of each diet was as follows: (1) Standard diet: A cholesterol restricted diet (Mayo Clinic" Diet Manual)
69 was used to provide baseline lipid conditions prior to other dietary studies. The older boy (age 8) received a diet consisting of 52 g protein, 224 g carbohydrate and 54 g t;at (30 35'/~,, lipid diet, 9 g saturated and 45 g polyunsaturated), total calories 1645. The younger boy (age 5) received 42 g protein, 188 g carbohydrate, 48 g fat (7 g saturated and 41 g polyunsaturated), total calories 1375 (as recommended by Dr. Wayne Callaway and Ms Huse, Department of Internal Medicine, Mayo Clinic). (2) Standard diet supplemented with 18. 2~o6: 2 g of 701~, ethyl linoleate (isolated from hog liver by NuChek, Elysian, MN) from freshly opened vials, sealed under nitrogen, was blended into food and drink and eaten directly after preparation to minimize fatty acid oxidation. (3) Standard diet supplemented with 18 : 3o~6 : 450 mg of evening primrose oil (EPO) containing 9')~ of 18 • 3co6 was supplemented. Since EPO is a seed oil, its use here was that of a food. (4) Standard diet supplemented with 20 . 3co6 : A naturally enriched source of 20 : 3~o6(dihomo-7-1inolenic acid or DHLA) was not available. Therefore, synthetic DHLA (Hoffman-LaRoche, UK) was substituted. A limited clinical trial of DHLA had been performed by Hoffman-LaRoche company (Stone et al. 1979) in the United Kingdom, but not in the U.S.A. Thus, special approval from the Food and Drug Administration, U,S.A., was obtained for this dietary trial (IND # 18079). The dosage of D H L A for each patient was as follows: 1 capsule (50 mg DHLA containing 0.1',}o butylated hydroxyanisole and 0.I"~i butylated hydroxytoluene as anti-oxidants) at breakfast daily for the first 2 weeks, 1 capsule at breakfast and supper daily for the following 2 weeks and 2 capsules at breakfast and supper and 1 capsule at lunch daily for the last 2 weeks. (5) Standard diet supplemented with 20 : 4co6" 2 g of 70~ ethyl arachidonate (isolated from hog liver by NuChek) was supplemented as in the standard diet with 18 : 2~o6. Lipid analysis The plasma (overnight fast) lipid and fatty acid compositions were analyzed on 2 or 3 consecutive days before and after each dietary trial. Lipid extraction from total plasma was performed according to the procedure of Nelson (1975). Nonpolar lipid subclasses and total phospholipids were separated from plasma lipid extracts by thin-layer chromatography (TLC) using hexane/diethyl ether/acetic acid (112: 35"2.5, v/v/v) as the developing solvent system (Yao et al. 1976). Following separation by TLC, a quantitative analysis of cholesteryl ester, triacylglycerol, free fatty acid, cholesterol and phospholipids was performed using a modified liquid scintillation counting procedure (Shand and Noble 1980). The TLC plate was first charred at 180°C for 15 min after spraying with 3% (w/v) cupric acetate in 8°o (w/v) phosphoric acid (Fewster et al. 1969). The charred bands were scraped off into scintillation vials and then suspended in 4 ml of distilled water. After the addition of 10 ml of Ready-Solv MP (Beckman, Irvine, CA 92713) scintillation cocktail, the vials were shaken vigorously to trap the charred lipids in the resultant
70 firm gel. The amount of quenching due to the presence of the charred lipids was measured by counting each sample for 5 rain in the external standard channels ratio (ESCR) mode of the liquid scintillation counter (Model LS-333. Beckman. Fullerton. CA 92634). The relationship between the ESCR and mass was linear up to at least 50/~g for all of the common lipid classes. Cholesteryl ester and triacylglycerol were transesterified using sodium methoxide reagent (Luddy et al. 1960~. Free fatty acid. phosphatidylcholine and total phospholipids were methylated with boron triftuoride/methanol reagent according to the method of Morrison and Smith (1964). The resulting fatty acid methyl esters were further purified by TLC. using petroleum ether/diethyl ether/acetic acid ( 9 0 : 1 0 : [ . v/v/vl as the developing solvent system (Metcalfet al. 1966). All methylated samples were analyzed on a Packard Gas Chromatograph model 419, equipped with dual hydrogen detectors. The operating conditions for gas-liquid chromatography (GLC) were as described previously (Yao et al. 1976~. Peaks on the chromatograms were identified by comparing the retention times with those of a variety of reference standard mixtures (Supelco. Inc.. Bellefonte, PA) and individual standards (Nu Check Prep. Elysian. MN and Analabs. Inc., North Haven. CT) and were calculated using the Autolab System IV computing integrator.
Follow-up evaluation The patients were medically assessed at weekly intervals throughout the study by (a) physical examination: (b) hemoglobin, leucocyte count and differential: (c) chemistry group; (d) complete urinalysis and (el chest X-ray. No attempt was made to ascertain clinical improvement because of the short treatment period. We were testing for biochemical restoranon only. RESULTS
The concentrations of the major plasma lipid subclasses after dietary trials are shown in Table 1. Among all the diets tested, only the standard diet-enriched in ethyl arachidonate substantially increased the plasma lipid concentration of both patients, particularly cholesterol and phospholipids. The percentage distribution of cholesteryl ester to total cholesterol remained essentially unchanged throughout the period of dietary trials. The ethyl arachidonate-enriched diet not only increased the arachidonate (20:40)6) content of plasma phosphatidylcholine for both patients (Fig. t), but also increased the proportion of 20:40)6 to total fatty acids 2.5-4.8-tbld in both cholesteryl ester and phospholipid fractions (Table 2). Concomitantly, there was a sharp decrease of 18 : 20)6 in both fractions. The proportion of 22 : ~06 in plasma phospholipids was slightly increased while that of 20 : 3o6 remained unchanged. The evening primrose oil-enriched diet produced no apparent increase of 18 : 30)6 in plasma phosphotipids (Table 3t. However. the ratio of20:3a,'6~18 : 3a,'6 was found to be substantially higher in both patients after the standard diet enriched with evenmg primrose oil compared with the standard diet alone.
FABLE 1 EI:EECT OF VARIOUS P O L Y U N S A T U R A T E D FATTY A C I D - E N R I C H E D DIETS ON PLASMA LIPID C O N C E N T R A T I O N S
Plasma lipids
Subjecl
Standard diet
Standard diet plus Ethyl linoleate
Phospholipid
111-7 111-6
Cholesterol
I11-7 II1-6
Triacylglycerol
Free fatty acid
85 _+ 3" 89 + 6
Evening primrose oil
Dihomo-> linolenate
Ethyl arachidonale
103 h 107
79 80
99 70
132 117
152 _+ 9 149 + 8
178 161
136 114
215 113
256 194
111-7 III-6
60 + 2 46 _+ 7
78 67
55 34
83 31
80 65
111-7 111-6
22 + 2 20 _+4
17 19
18 20
25 19
27 29
:' Mean value Img/dl) and SD of 3 samples obtained from patients on 3 different occasions after a standard diet. h Mean value of 2 samples obtained on 2 consecutive days after designated diet.
14
.
.
.
.
121-7
B
4
2
8 7 6 5 4 5 2 1 0
18:2 20:3 20"4 ~o~ P O L Y E N O I C FATTY ACIDS = Standard diet = Standard diet enriched in ethyl araehidonate = Standard diet enriched in d i h o m o - ~ q i n o l e n a t e
Fig. l. Effect of standard diet supplemented with either ethyl arachidonate or dihomo-7-1inolenatc (DHLA~ on 18 : 2uJ6, 20:30)6 and 20 : &o6 contents of plasma phosphatidylcholine. Phospholipid fatty acids were quantified by gas chromatography using methyl pentadecanoate as an internal standard. Each bar represents the mean of 2 or 3 samples obtained from patients on 2 or 3 consecutive days after a designated diet. 111-7 and 111-6 represent 2 affected patients.
72 After feeding both patients a DHLA-enriched diet. the proportion of 18 : 2co6 was elevated consistently in both free and esterified fatty acid fractions (Tabte 4J. This increase was accompanied by a decrease o f 18 : lco9. Furthermore. the ratio o f 20 : 4o96/20:3co6 in plasma lipids was suppressed to one half to one third of that control value (obtained after standard diet).
TABLE 2 E F F E C T OF L I N O L E A T E A N D A R A C H 1 D O N A T E - E N R t C H E D D IETS O N P L A S M A c~6 POLYENOIC FATTY ACIDS
Fatty acids
Standard diet
Cholesteryl
Standard diet plus Ethyl linoleatc
Ethyl a ra c hi dona t e
II1-"
1II-6
I[1-7
111-6
III-7
Il t -6
56.8 _* l . P 1 4.1 _ 0 . 1
56.5 z 1.4 4.4 z 0.2
61.1 ___ 1.7 4.1 ___0.4
58.9 -+ 2.2 4.3_+0.2
38.2 z 0.5 18.3 z 0.4
39.4 -+ 0.3 t5.1 z 0 . 3
24.5 ~- 0.2 3.2_+0.3 6.2-1-0.3 1.0 -+ 0.1
2 6 3 z 1.6 3.0_+0.1 7.5_*0.7 1.1 z 0 . 1
29.5 z 1.9 3.0 ~_ 0.4 6.6m 1.1 1.0 ~ 0.2
27.9 ~ 1.4 3.1 ___0.3 7.6-,-0.9 1.1 ~_0.1
14." m 1.6 2.8±0.1 20,6_+0.6 1.9 _T0.2
14.7 _+_1.4 2.9_+0.4 19.0-+1,] 1,8 ~_ 0.2
ester
18 : 2 20:4 Phospholipids
18 : 2 20:3 20:4 22:4
° Mean value (wt %) and SD of 3 samples obtained on 3 consecutive days. The saturated and m o n o u n s a t u r a t e d fatty a c i d s w e r e not included in tabulation.
TABLE 3 E F F E C T OF S T A N D A R D D I E T E N R I C H E D IN E V E N I N G P R I M R O S E O I L (EPO) O N (06 P O L Y E N O I C F A T T Y A C I D S OF P L A S M A P H O S P H O L I P I D S .
.
.
Fatty acids
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Standard diet -
18 : 2 18 : 3 20 : 3 20 : 4 20 : 3, 18 : 3
.
-
EPO
II1-7
11I-6
1II-7
111-6
24.62 a 0.35 2.89 5.54 8.26
25.46 0.38 2.24 6.76 5.89
24.36 0.29 3.56 6.04 12.28
25.82 0.32 3.05 7.27 9.53
Mean value (wt ~,(,) of 2 samples obtained on 2 consecutive days.
73 TABLE 4 EFFECT OF D I H O M O - 7 - L I N O L E N I C ACID (DHLA) DIET ON PLASMA FATTY ACID COMPOSITION
Fany acids ~'
Free fatty acids 111-7
16:0 18:0 18 : hJJ9 18 : 2oj6 20 : 3u06 20 : &o6 20 : 4eJ6,' 20 : 3c96
Cholesteryl ester 111-6
1II-7
Phospholipids 111-6
111-7
111-6
Std t,
DHLA
Std
DHLA
Std
DHLA
Std
DHLA
Std
DHLA
Std
DHLA
6.4" 12.8 49.5 23.4 0.6 0.8
6.2 13.2 34.2 35.5 1.3 1.6
16.1 11.8 45.9 19.3 0.5 0.5
19.1 7.9 28.2 33.7 0.5 0.6
7.8 1.4 21.0 62.1 0.5 3.8
8.9 0.8 15.3 68.7 1.0 3.1
6.1 0.7 17.8 67.7 0.5 4.4
6.2 0.7 12.4 75.2 0.9 3.3
27.4 14.1 16.8 30.6 2.2 4.3
27.1 14.3 9.8 34.3 4.7 4.6
21.6 14.6 15.2 ~5. ) 2.7 5.4
21.6 15.3 9.6 40.6 4.4 3.5
1.3
1.2
1.0
1.2
7.6
3,1
8.8
3.7
2.0
1.0
2.0
0.8
~' Minor fany acids, 14:0, 16: 1, 17:0, 18:3, 20:0, 20: 1, -~0 '~..~, 21:0, 22:4, ,,'~:5, ,_'~'~:6, 2 3 : 0 and 2 4 : 0 . were not tabulated. I~ Both patients (11I-7 and Ill-6) were placed on a standard (std) diet for 3 weeks just prior to the D H L A trial. L, Each value (wt <',,) represents the mean of 2 samples obtained on 2 consecutive days afier the designated diet.
DISCUSSION
These studies have shown that the low levels of plasma 20 : 4co6 encountered in our patients with multineuronal degeneration, hepatosplenomegaly and adrenal failure (Dyck et al. 1981) can be raised above normal using a diet enriched in ethyl arachidonate. No adverse clinical or laboratory findings were recognized, indicating that a long-duration trial of ethyl arachidonate may be possible. The marked increase of 20:&o6 was found at the expense of 18:2~o6 in both plasma cholesteryl ester and phospholipid fractions. A similar finding has been demonstrated in rat plasma after an ethyl arachidonate diet (Danon et al. 1975). This dramatic decrease in 18:2o)6 cannot be a simple dilution effect, but suggests that there is competitive incorporation between substrate and product in the ~o6 polyenoic fatty acid pathway. Arachidonate appears to be the preferred substrate for fatty acid esterification. Lack of accumulation of linoleate also suggests that there is no reverse conversion of arachidonate to linoleate (Mohrhauer and Holman 1963; Rahm and Holman 1964). We (Dyck et al. 1981) have previously shown that these patients have a normal plasma composition of 18 : 2~o6. Alter the standard diet was supplemented with ethyl linoleate, there was no apparent increase of any desaturated or elongated products further supporting the fact that these patients did not have a substrate deficiency in their cJ6 polyenoic fatty acid pathway to account for the decreased concentration of 20 : 4~o6. When 18 : 3~/J6 was fed to rats, no significant amounts of this acid could be detected in their livers (Rahm and Holman 1964; Sprecher 1974). The 18:3~o6
74 was rapidly elongated to 2 0 3~96 (Sprecher 1977). An enhanced, but insignificant. level of 18 • 3a~6 was found in the liver when 18 • 3co6 was fed to rats with essential fatty acid deficiency (Garcia and H o l m a n 1965). Therefore. lack of incorporation of 18 " 30,'6 into plasma lipids in these two patients, after a standard diet supplemented with evening primrose oil (Table 3), was probably due to a rapid elot~gation of 18 • 3o,'6 to 20 : 3co6. An increased ratio of 2 0 3co6J18 • 30,,6 (Table 3) supports this idea. Because the p r o p o r u o n of 20" 3~o6 generally is found to be much lower m mammalian tissue than that of 20 : 4v06. the principal biosynthetic role of 20 : 3~J6 is considered to be that of a precursor for 20:4eo6 (Sprecher 1977). Therefore. it ~s not surprising to find a low incorporation of 20 : 3~o6 in these two patients after a standard diet supplemented with D H L A Lack of increase of plasma 20 : 4e~6 by supplements of either ethyl linoleate or D H L A suggests that a defect of A5 desaturase may be involved. On the other hand, there was an increase of both free and esterified plasma linoteate (18"2eo6) content after the D H L A - e n r i c h e d diet (Fig. l and Table 4). indicating that a retroconversion of 20:3a~6 to 18:2co6 is active in these two patients. Whether this retroconversion is also present in healthy subjects is unknown. However. when either D H L A ester or D H L A was administered to rats or rabbits. the level of plasma linoleate of cholesteryl ester and phospholipids was significantly reduced (Danon et al. 1975 Oelz et al. 1976: Stone et al. 1979). Therefore. a retroconversion of20:3~o6 to 18 - 2c06 may be a key step in limiting A5 desaturasc activity in these two patients. In fact, various evidence (Verdino et al. 1964: Sprecher 1967. 1977) supports the hypothesis that termination ot" polyunsaturated fatty acid biosynthesis ~s regulated by a balance between chain elongation and desaturation reactions versus retroconversion. We have also reported a small, but unequivocally elevated amount of branchedchain fatty acids, phytanic and pristanic acids, in these two patients' plasma fYao et al. 1982). Their presence was not effected by dietary supplementation of various a~6 potyenoic fatty acids. REFERENCES Baker. R.W.. R. H. T h o m p s o n and K. J. Zilkha {19641 Serum f a n y acids in multiple scler~si,..I. Neurol. Neurosurg. Pa3'ehiat.. 27:408 414, Collins, F.D.. A.J. Sinclair. J.P. Royle, D . A , Coats, A . D Maynard and R . F . L c o n a r d (1971) Plasma lipids in h u m a n linoleic acid deficiency, Nutr. Metab., 13: 150--167. Danon. A.. M. Heinberg and J.A. Oates (1975] Enrichment of vat tissue lipids with fatty acids that are prostaglandin precursors, Bioehim. Biophys. Acta, 388:318 330, Davigon. J.. Y.S. Huang, J.P. Wolf and A Barbeau (1979) Fatty acid profile of major lipid classes m plasma lipoproteins of patients with Friedreich's ataxia Demonstration o f low tinoleic acid content most evident in the cholesterol-ester fraction. Can. J. Neurol, Sci., 6 : 2 7 5 293 Dyck. P.J.. J. K Yao. D . E Knickerbocker, R . T . Hotman, M . R . Gomez, A, B. Hayles and E.H. Lambert t198t) Multisystem neuronal degeneration, hepatosplenomegaly, and adrenoconical deficiency associated with reduced tissue arachidonic acid. Neurology (Minneap,}. 31 : 925 934. Fewster. M.E. B,J. Burns and J.F. Mead (1969] Q u a n m a t i v e densitometric thin-layer chromalography of lipids using copper acetate reagent. J. Chromatogr., 43: 120-126.
75 (iarcia, P . I . and R.T. Holman (1965) Competitive inhibitions in the metabolism of polyunsaturated fatty acids studied via the composition of phospholipids, triglycerides and didacteryl esters of rat tissues, J. Amer. Oil Chem. Sue., 42: [ 137 1141. Holman. R.T. (1970) Biological activities of and requirements for polyunsaturated acid. In: R.T. Holman (Ed.). l'ro~,,rcss m the Clwnfistrl o/ t.~IL~ and O/her Lipi&', l'o/. 9, Pergamon Press Ltd., Oxlord. pp. 606 682. Kuo. P.T. and N.N. Huang (1965) The tit"cot of medium chain triglyceride upon fat absorption and plasma lipid and deposition of fat in children with cystic fibrosis of the pancreas, J. ('li~t. hn'esl., 44:1924 1933. Kuo. P T . . N.N. Huang and D. R. Bassett [196l) the laity acid composition of the scram chylomicrons and adipose tissue of children wilh cystic fibrosis of the pancreas..I. Pcdkl/.. 60:394 403. I.Ioyd-Sli[l, ,1.1)., S. B..Iohnson and R.T. Holman (1981) Essential Iatty acid status in cystic fibrosis and the effects of safflower oil supplementation, Amer, J. ('lhl. Nulr., 34:1 7. Love, W.C., A. Cashell, M. Reynolds and N. Callaghan (1974) Linoleate and lhtty acid patterns of serum lipids m multiple sclerosis and other diseases, Brit. Med. J., 111:18 21. Luddy. F.E., R.A. Barford and R.W. Riemenschneider (1960) Direct conversion of lipid components to their larry acid methyl esters, J. Miner. Oil ('lwm., 37:447 451. Metcalf, L.D.. A.A. Schmitz and J.R. Polka (1966) Rapid preparation of fatty acid esters from lipids Ior gas chromatographic analysis, 4mtlrt. Chem., 38:514 515. Mohrhauer, H. and R.T. Holman (1963) Effects of dietary essential fatty acids upon polyunsaturated lhtty acids m rat heart tissue. In: A.C. Frajer (Ed.), Biochemistry Prohlems O( LipkL~', Elsevier Publishing Company. Amsterdam, pp. 446 452. Morrison, W.R. and L.M. Smith (1964) Preparation of fatty acid methyl esters and dimethyIacetals froln lipids with boron lluoride methanol. J. Lipid Re,s., 5:61)0 608. Nelson. G.J. (1975) Isolation and purification of lipids from animal tissues. In: E.G. Perkins (Ed.), .4mt@s'i.~ O~ Lipi& and Lipoproteh~s, American Oil Chemists" Society, Champaign, IL, pp. 1 22. Oelz, O.. H.W. Seyberth. H.R. Knapp, B.J. Sweetman and J.A. Oates (1976) Effects of Ik'eding cthyl-dihomo-;'-linolenatc on prostaglandin biosynthesis and platelet aggregation in the rabbit, Bi(whint. BIol)h.vs. .4eta, 431:268 277. Rahm. J.J. and R.T. Holman (1964) Studies of the metabolism of polyunsaturated acids by shortterm expcrimcnts. J. Nutr.,84:149 154. Shand, H. and R.('. Noble /1980) Quanlification of lipid mass by a liquid scintillation counter procedure I'ollouing charring on thin-layer plates, Anahv. Biochem.. 101: 427-434. Sprecher. H. (1967) ]"he total synthesis and metabolism of 7,10,13,16-docosatetracnoate in the rat, Biochinl. Biophy~. At/a, 144:296 304. Sprecher, H. (1977) Biosynthesis of polyunsaturated fatty acids and its regulation. In: W.-H. Kunau and R. T. Holman (Eds.), Polvunsaturawd l,'atly Act&, American Oil Chemists" Society, Champaign. 11., pp. l 18. Spritz, N. and M.A. M ishkel (1969) Effects of dietary Ihts on plasma lipids and lipoproteins An hypothesis fbr the lipid-lowering effect of unsaturated fatty acids, J. Clin. lnvesh. 48: 78-86. Stone, K.J.. A.L. Willis, M. Hart. J.J. Kirtland, P.B.A. Kcrnoff and G.P. McNicol (1979) ]"he metabolism ot'dihomo-i'-linolenic acid in man, Lipi&, 14: 174-180. Verdino, B., M.L. Blank. O.S. Privett and W.O. Lundberg (1964) Metabolism of 4,7.10,13,16docosapentaenoic acid in the essential fatty acid-deficient rat, J. Nutr.. 83:234 238. Yao. J.K. and P.J. Dyck (1978) Lipid abnormalities in hereditary neuropathy, Part 2 (Serum phospholipids), J. Nem'ol. Sci., 36:225 236. Yao, J. K., R. D. Elle£'~on and P.J. Dyck (1976) Lipid abnormalities in hereditary neuropathy. Part 1 iScrum non-polar lipids), J. Neuro/. Sci., 29:161 175. Yao. J.K., 1. Jardine and P.J. Dyck (1982) Presence of plasma branched-chain l'atty acids in multineuronal degeneration, hepatosplenomcguly and adrcnocortical insufficiency, ,I. Neurol. Sci., 55: 185 195.