Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney S. Skrede & O. Slaattelid To cite this article: S. Skrede & O. Slaattelid (1979) Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney, Scandinavian Journal of Clinical and Laboratory Investigation, 39:8, 765-771, DOI: 10.1080/00365517909108169 To link to this article: http://dx.doi.org/10.1080/00365517909108169
Published online: 14 Feb 2011.
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Scand. J. clin. Lab. Invest. 39, 765-71 1, 1979.
Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney
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S . S K R E D E & 0. S L A A T T E L I D Institute for Surgical Research, Surgical Department B and Institute of Clinical Biochemistry, Rikshospitalet, National Hospital of Norway, University Hospital, Oslo
Skrede, S. & Slaattelid, 0. Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney. Scand. J. clin. Lab. Invest. 39, 765-771, 1979. Perfusion of isolated dog kidneys was performed at 8-12°C using an albumin solution containing caprylic acid (about 6 mmol/l) and long-chain fatty acids (about 0.5 mmol/l). During 48 h the amounts of caprylic acid in the perfusate fell by about 3 mmol, whereas long-chain FFA increased by 0.2-0.3 mmol and small amounts of arachidonic acid appeared. [I4C]palmitate or [ 14C]linoleate-when added-decreased by about 10% in the perfusate. The decrease was mainly due to exchange with kidney phospholipid fatty acids. Only about 0.4% was recovered as [14C]C02.The amounts of total phospholipids in kidney tissue decreased by up to 10% during the perfusion (when 10% kidney weight gain was taken into account), whereas lysophospholipids, cholesterol, triglycerides and free fatty acids remained essentially unchanged. The distribution of fatty acids in the total phospholipid fraction was strikingly altered. The relative amounts of arachidonic acid increased, whereas all other major fatty acids decreased. Synthesis of arachidonic acid by chain elongation of perfusate [14C]linoleicacid could not be demonstrated, and no evidence was found for a significant increase of the total amounts of arachidonic acid. The possibility that arachidonic acid-containing phospholipids in the kidney are preferentially preserved during the hypothermic conditions is discussed. Key-words: arachidonic acid; fatty acid metabolism; hypothermic perfusion; kidney preservation; phospholipid fatty acids S . Skrede, M.D., Institute of Clinical Biochemistry, Rikshospitalet, Pilestredet 32, Oslo 1
In a previous study [22] we have found that the functional capacity of dog kidneys, preserved for 48 h by hypothermic perfusion, was better when free fatty acids (FFA) were present in the albumin perfusate. The concentration of total FFA in the perfusate decreased during perfusion. 0036-551 3/79/1200-0765902.00
0 1979 Medisinsk Fysiologisk Forenings Forlag
The disappearance of radiolabelled palmitic acid was rather low, however, and the results suggested that other fatty acids, e.g. caprylic acid, were removed at a higher rate than palmitic acid. In the present study, the fate of individual perfusate fatty acids during kidney perfusion has been investigated. The amounts of renal tissue lipids and the phospholipid fatty acid 765
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pattern before and after perfusion were also studied. MATERIAL A N D METHODS
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Reagents. The basis for the perfusion fluid was a 20% human serum albumin solution, obtained from KABI AB, Stockholm, Sweden. [I4C(U)]palmitic acid and [ 14C(U)]linoleic acid were purchased from New England Nuclear, Boston, Mass. USA. All other reagents were commercial products of high purity. Atiimnls and surgical procedures. Six mongrel dogs ( I S 1 8 kg) (exp. a-f) were used in this study. They were fasted overnight before the operative procedure, but had free access to water. The dogs underwent bilateral nephrectomy under pentobarbitone anaesthesia (Nembutal veterinary, Abott Laboratories Ltd, Chicago, Ill., USA). The dose was 30 mg/kg body weight. The right kidney was removed first and served as a control. After initial flushing [22] the kidney was weighed and placed in icecold isotonic saline. The kidney was decapsulated and we took two to four wedge-shaped biopsies (dissected free of fat); we intended to obtain mainly cortical tissue (the amount of medullary tissue was probably always less than 5 04). After weighing, the biopsies were frozen. The left kidney was then dissected free, the vessels were clamped and cut and the kidney removed. Kidney preservation and perjusiotz niedia. Immediately after removal, the left kidney was flushed, weighed and then perfused for 48 h in the Gambro perfusion machine (Gambro, Lund, Sweden) as previously described [22]. The machine was primed with a volume of 850 ml (in experiments b, e and f : 600 ml). The perfusate consisted of fatty acid-containing albumin with additives [22]. The perfusate did not contain triglycerides or cholesterol. In two experiments ['4C]palmitic acid or [14C]linoleic acid, (50 pCi) complexed with serum albumin [19] was added to the perfusate. In these experiments a C02-trap with 30% KOH was connected to the outlet of the oxygenator. After preservation, the kidney was weighed and then treated in the same manner as the right (control) kidney. During perfusion for 48 h a
gain in kidney weight was always present, and averaged 10%. Perfusate samplitig. Before perfusion started and then with intervals, perfusate samples were taken for determination of free fatty acids. Perfusate radioactivity was measured before and after kidney perfusion. When calculating the recovery of 14C-labelled fatty acids, the amounts recovered by the flush-out were also included. Samples of the perfusate were taken for sterile control. No bacterial contamination was found. Biochemical methods. 'Total free fatty acids' (mainly long-chain fatty acids) were estimated using a titrimetric method introduced by Dole [7] and modified by Trout et al. [23]. The total amounts and the relative distribution of longchain fatty acids were also determined by gas liquid chromatography (GLC) [8] (esterified fatty acids were not detected in the perfusate). Folch-extracts [9] were prepared in duplicate, and long-chain fatty acids were estimated after methylation with pentadecanoic acid as internal standard, using an 8 % BDS-column at 180°C. Caprylic acid was determined after deproteinization with methanol, acidification, extraction with diethylether and methylation, with nonanoic (pelargonic) acid as internal standard [ 121. A n 82, BDS-column was used at 110°C. The coefficients of variation for the quantitation by GLC of the individual long-chain fatty acids as calculated from nineteen duplicate perfusate analyses (including the extraction step) were: 16:O 3.4%; 61:l 9.3%; 18:O 11.3%; 18:l 4.5%; 18:2 4.4% and 20:4 (nine duplicates, after perfusion only) 7.4%. The total lipids in the perfusate as well as in the renal tissue were extracted according to Folch [9]. The recovery of [14C]palmitate from the perfusate was about 95%. In aliquots of the renal tissue lipid extract, cholesterol was estimated according to Levine & Zak [16], and triglycerides according to Kessler & Lederer [13]. Phospholipids were estimated according to Zilversmit [25]. Total phosphorous was estimated by the same method, but starting with the combustion step. Thin-layer chromatographic separation of phospholipids and cholesterol was achieved using silica gel H with a solvent mixture of petrol ether (b.p. 4O-6O0C), diethylether: acetic acid (85:15:5, v/v) [lo]. For the separation of triglycerides and free fatty acids,
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Kidney lipids during hypothermic perfusion the solvent system was hexane: diethyl-ether: acetic acid (80:20: 1, v/v). When estimating 14C-activity, the zones were visualized in an iodine vapour chamber, scraped into glass scinter funnels and repeatedly eluted using chloroform/methanol (2: 1). The phospholipid zone was in addition eluted with methanol. I4C-activity was estimated by liquid-scintillation. For quantitative lipid analyses, only standards on the lateral areas of the thin-layer plates were sprayed with 0.20,/,fluorescein. Lysolecithin and lecithin were separated by thin-layer chromatography on silica gel H, using a solvent mixture of chloroform: methano1:water:acetic acid (65: 35:5:4 v/v). Egg lecithin and lysolecithin were used as standards. The fractions were localized by spraying with 0.2% fluorescein in ethanol and eluted according to Arvidson [I]. Phospholipids were then combusted, and inorganic phosphate estimated according to Bartlett [2]. The total phospholipids were hydrolysed at 80°C for 40 min. with ethanol/KOH (30%), and the content of long-chain fatty acids was estimated as for perfusate fatty acids (see above). Caprylic acid was not detected in this fraction. A combined GLC/MC instrumentVarian MAT 112 (Varian MAT, Bremen, Western Germany) was used to confirm the structure of the peaks (also 20:4). In the experiment when [ ‘T]linoleic acid was present in the perfusate, radio-gas-chromatography of the phospholipid fatty acids were performed with a Hewlett-Packard instrument using a SB 2340 column at 200°C.
RESULTS Perfusate analyses Table I shows the results of Dole titration (‘total fatty acids’, [7, 231) at different intervals during kidney perfusion, and also the concentration of individual fatty acids as estimated by GLC. In accordance with previous results [22], a gradual decrease of the ‘Dole titre’ was found. By GLC analyses, a high concentration of caprylic acid was found in the perfusate (this medium chain fatty acid is added to the albumin solution). Table I also shows that the total fatty acids in the perfusate are underestimated by Dole’s method [7, 231, since caprylic acid is estimated with a recovery of only 50-60% by this method (own results, which agree with those of Linscheer et al. [17]). Table I shows that the decrease of caprylic acid during the perfusion is of such an extent that it explains that the ‘Dole titre’ decreases, even though the longchain fatty acids in fact increase. GLC analyses showed that the conccntration of all long-chain fatty acids increased by on an average 60% during 48 h in four experiments, and also that small amounts of arachidonic acid appear in the perfusate in this period. The total phosphorous content of the perfusate increased by about 0.4 mmol during 48 h (exp. e, results not shown). Figure 1 shows the time-course of the increase of the different long-chain fatty acids during the first 8 h of perfusion. Arachidonic acid appeared rather rapidly (after 2 h), and the increase of the
TABLE I . Changes of the fatty acid pattern of the perfusate during kidney perfusion at 10°C for 48 h . (All analyses were performed in duplicate, including the extraction step-cf. Methods.) Perfusion ‘Dole time titre’* Experiment (h) (pmol/l) a b
0
C
d C
d a b
24 48
C
d
* Cf.ref. (23), ** N.D.:
Perfusate concentration (pmol/l) 8:O
16:O
16:l
18:O
18:l
18:2
20:4
2860 3690 2740 2980
5790 6630
30
-
28 31 27 33 34 40 46
50 67 57 44 74 39
188 174 155 144 198 216 283 286 22t 283
103 126 96 88 100
N.D.** N.D. N.D. N.D.
-
181 204 156 144 197 216 272 400 218 273
100
-
129 184 107 116
27 53 17 16
2343 2040 2870 2200 1880
-*** -
2230 3350
-
~
Not detectable,
767
*** -:
36
43
100
127 70 84
Not performed
~
168
S. Skrede & 0.Slaattelid
Tissue analyses
P
T
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C 0 0
Y
U 0
x c +
8
4 Time i h )
FIG.1 . Increase of long-chain fatty acids in the perfusate during the first 8 h of kidney perfusion (exp. e). Lipid extractions according to Folch e t a / . [9] were performed in duplicate, and from each lipid extract the analyses (cf. Methods) also proceeded as duplicates. The vertical bars indicate the ‘overall SD’ for these four measurements. The symbols for the different fatty acids are: I! 16:O; 16:l; 1 8 : O ; 0 1 8 : l : 0 18:2; A 20:4
other fatty acids (e.g. 16:O) was almost linear during the first 8 h. A continued gradual increase in the level of different fatty acids is shown by Table 1. In the experiments when [‘4C]palmitic acid or [14C]Iinoleicacid was added to the perfusate, a reduction by 10% of perfusate 14C-activity was recorded during 48 h of kidney perfusion (Table 11). This I4C-activity was almost completely recovered from lipid extracts of the perfused kidney. Only traces of I4C-activity (0.36% of total radioactivity) was recovered from trapped CO,.
The amounts of phospholipids (and lysophospholipids), total cholesterol, triglycerides and free fatty acids in control and perfused kidney tissues are shown by Table 111. When it is born in mind that the wet weight of the kidney increased about loo/,, it is seen that the total phospholipids decreased by up to 10%. The lysolecithin fraction was not increased. Total cholesterol was not significantly changed during 48 h perfusion, when the precision of the method and the kidney weight increase is taken into account. The amounts of triglycerides (with the exception of experiment b) and free fatty acids were small, and were not significantly changed (even a complete wash-out of the kidney free fatty acid pool would have contributed to a minor part of the observed increase of fatty acids in the perfusate). Table IT shows that I”C-activity from palmitic acid or linoleic acid was incorporated into tissue lipids. The incorporation into the phospholipids was at a considerably higher level (more than 6% of total initial perfusate activity) than in the other lipid fractions. Before perfusion, the amounts of kidney phospholipid fatty acids was estimated to be 40-50 pmol/g wet weight. After 48 h perfusion, the amount per g wet weight decreased by on an average 15-20% (when no corrections for about 10% wet weight increase were performed). Table IV shows that the relative distribution of different phospholipid fatty acids had changed in this period. Arachidonic acid underwent a relative increase at the expense of most of the other individual fatty acids. We found no evidence for an increase of the total amounts of this fatty acid, when the precision of the methods for kidney wet weight determination and total phospholipids were taken into account. The
TABLE I I . Recovery of “C-activity after 48-h kidney perfusion with [“Clpalmitate (a) or [‘4C]linoleate (b) i n the perfusate ‘‘C-activity present in kidney lipids (% of initial [14C]palrnitate or [“C]linoleate activity in the perfusate) Time of perfusion Exp. (h) a b
48 48
Perfusate 90.9 88.5
Total extract Free C02-trap of tissue lipids Phospholipids cholesterol Triglycerides FFA 0.36 ~
8.6 12.5
6.2 6.4
0.3 0.3
0.1 0.7
1.5 5.1
Kidney lipids during hypothermic perfusion
769
TABLE 111. Pattern of the major lipid classes in the renal tissue before and after 48-h kidney perfusion
Time of perfusion Experiment (h) a
0 48 0 48 0 48
b e
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Amounts of tissue lipid (,umol/g wet weight) Kidney weight Phospholipids Total Free fatty acids (9) Total Lysolecithin cholesterol Triglycerides (sum of long-chain
* Two control
81.5 87.8 56.5 58.1 69.0 76.5
32.0 25.2 26.3 22.8 27.8 21.9
0.25 0.25 -
__ -
8.0* 7.9* 8.6 8.9
0.7 0.6 5.0 5.5 0.4 0.4
-)
I .o 0.9
kidney samples and two perfused kidney samples were analysed in quadruplicates (CV =
2%).
TABLEI V . Changes of the fatty acid pattern i n renal phsopholipids during 48-h kidney perfusion Timeof perfusion Experiment (h) a
b e f a b
0
48
No. of tissue samples
Percental distribution of major fatty acids*
2
8:O N.D.**
1
N.D.
2
N.D.
I 3
N.D. N.D.
1
N.D.
16:O 18:O 18:1 18:2 20:4 30.57 14.8 22.4 14.9 17.5 (26.8-33.1 1 (14.3-15.6) (21.6-23.8) (14.2-15.4) (16.0-19.2) 28.0 13.9 19.7 16.3 22.1 26.1 14.2 20.6 17.7 20.9 28.2 14.1 18.7 18.3 20.7 28.5; 13.5 27.1 19.2 11.5 (28.1-30.1) (13.3-13.8) (19.1-19.2) ( 1 1.1-11.9) (26.2-28.4) 26.5 12.2 17.4 17.7 26.2
* Additional minor peaks (e.g. palmitoleic acid (16:1) and unidentified peaks) always contributed with less than 3-4%, and were not included in the sum. ** Not detectable. 'f Mean of duplicate analyses (range in parentheses). Mean of three single analyses (range in parentheses).
amounts of all other fatty acids decreased, however. The composition of the kidney free fattyacidpool(l6:O 40%; 18:020%; 18:120% and 20:4 8%) did not change significantly during 48 h perfusion. In accordance with the previous study on the metabolism of [ 14C]caprylate [20], significant amounts of caprylic acid were not detected in the phospholipids of the perfused kidney by GLC/ MS-methods. In the experiment with ['4C]linoleic acid (exp. b), no incorporation of radioactivity into renal phospholipid arachidonic acid could be demonstrated by radio-gas-chromatography. (The sensitivity of the system would allow detection of a total kidney synthesis of as little as 0.3 pmol of 20:4 during 48 h.)
DISCUSSION In a previous study [22] we found that fatty acids bound to the perfusate albumin were important for the maintenance of kidney function during the preservation period. This widely used albumin-based perfusate contains caprylic acid at a high concentration (about 5-6 mmol/l) and the same long-chain fatty acids as in normal serum in about the same concentrations (sum about 0.5-0.6 mmol/l). In agreement with previous results [20], the present study shows that caprylic acid in the perfusate is rapidly utilized (by oxidation) by the dog kidney during hypothermic perfusion. Palmitic acid, on the other hand, was oxidized to COz at a very low rate.
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As observed previously [ l l , 201, long-chain fatty acids (e.g. [14C]palmiticor fl"C]linoleate) were incorporated into tissue phospholipids, and to a lower extent into kidney triglycerides. In the free cholesterol fraction, traces of I4C from palmitic acid of linoleic acid were recovered, probably by de iiow synthesis of cholesterol utilizing acetate generated by breakdown of the fatty acid. Kidney total phospholipids decreased by up to lox, without an increase in the lysophospholipids. Concomitantly, long-chain fatty acids (and total phosphorous) in the perfusate increased, and arachidonic acid also appeared. Previously Pettersson et a/. [20] did not find this decrease of the tissue phospholipids using essentially the same conditions for perfusion as we did, whereas Huang et al. [I 11 found that phospholipids were lost during kidney perfusion with cryoprecipitated plasma perfusate. Our finding that long-chain fatty acids in the perfusate increased during the perfusion is at variance with the report of Pettersson et al. [20], who also stated very high concentrations of individual fatty acids before perfusion (e.g. 18:l at almost 2000 pmol/l). The analytical performance of the quantitative gas chromatographic analyses of long-chain fatty acids in the present study has been documented (cf. Material and methods). The decrease of kidney phospholipids evidently reflects a changed balance between catabolism and synthesis. Possibly, de nouo phospholipid synthesis becomes reduced because of a loss of cytidine nucleotides, although Collste [6] did not find great changes of the total adenine nucleotide pool during kidney perfusion. I t is well known that when the temperature is lowered, e.g. in hibernating mammals [15] as well as in lower organisms [18], the phospholipids will contain a greater amount of unsaturated fatty acids. Changes in the pattern of long-chain fatty acids in the phospholipid fraction during hypothermic kidney perfusion have not been reported previously. We found a fatty acid pattern prior to perfusion which was in accordance with previous studies [5]. During 48 h perfusion, however, the relative distribution of fatty acids changed remarkably, with a great relative increase of arachidonic acid. The kidney has efficient fatty acid chain elonga-
tion mechanisms [3], but we did not find 14Cincorporation into arachidonic acid from perfusate [14C]linoleic acid. The cause of the changed distribution was that no significant changes in arachidonic acid were present, while all other fatty acids decreased. Arachidonic acid and other unsaturated fatty acids are predominantly found in the 2position of the phospholipids, which is more 'metabolically active' than the 1-position (241. The fatty acid in the 2-position is subjected to a cycle of deacylation (by phospholipase A l ) and a reacylation (acyl-CoA: lysolecithin acyl-transferase [14]. The present study does not give evidence to show whether a slow deacylation of arachidonylic species occurred, or whether a rapid reacylation of this fatty acid took place. In favour of the latter mechanism is that acylCoA : lysolecithin acyl-transferase preferentially reincorporates unsaturated fatty acids [ 141, and that acyl-CoA hydrolase is more active with short-chain unsaturated fatty acids [14]. The chainlength of the fatty acid may also be of importance for the reincorporation, since arachidonic acid is incorporated more rapidly than oleic acid by this mechanism [4]. In relation to our observations, it is interesting that in E. coli, the acyl-transferase is relatively more active with the unsaturated fatty acids than with the saturated ones when the temperature is lowered [l8, 211. This may explain the greater content of unsaturated fatty acids in the phospholipid fraction of bacteria at low temperatures. A similar temperaturedependence may be the reason for the preservation of arachidonic acid in the phospholipid fraction of the mammal kidney during hypothermic preservation. Other mechanisms are also possible, e.g. that phospholipase A l is relatively less active at lower temperatures against phospholipids containing arachidonic acid. ACKNOWLEDGMENT The excellent technical assistance of Ms Anne Marie Lund is gratefully acknowledged. REFERENCES 1 Arvidson, G.A.E. Structural and metabolic hetero-
genity of rat liver glycerophosphatides. Eur. J. Biochem. 4, 478, 1968.
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Kidney lipids during hypothermic perfusion 2 Bartlett, G.R. Phosphorus assay in column chromatography. J . biol. Chem. 234, 466, 1959. 3 Bojesen, I., Bojesen, E. & Capito, K. In vitro and in vivo synthesis of long-chain fatty acids from (I-'4C)acetate in the renal papillae of rats. Biochint. brophys. Acta 424, 8, 1976. 4 Brandt, A.E. & Lands, W.E.M. The effect of acylgroup composition on the rate of acyl transferasecatalyzed synthesis of lecithin. Biuchini. biopltys. Acta 144, 605, 1967. 5 Butkus, A., Robertson Lazzarini, A., Ehrhart, L.A. & Lewis, L. Renal lipids of dogs fed on arteriosclerosis-inducing diet. Exp. molec. Path. 17, 55, 1972. 6 Collste, H. Preservation of kidneys for transplantation. Experimental studies. Acta chir. scanrl. Suppl. 425, 1972. 7 Dole, V.P. A relation between non-esterified fatty acids in plasma and the metabolism of glucose. J . clin. Inivst. 35, 150, 1956. 8 Eldjarn, L., Try, K . , Stokke, O., Munthe-Kaas, A.W., Refsum, S., Steinberg, D., Avigan, J. & Mire, C. Dietary effects on serum-phytanic-acid levels and on clinical manifestations in heredopathia atactica polyneuritiformis. Lancet i, 691, 1966. 9 Folch, J., Lees, M. & Sloane Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. f. b i d . Chem. 226, 497, 1957. 10 Gloster, J . & Fletcher, R.F. Quantitative analysis of serum lipids with thin-layer chromatography. Clin. chim. Acta 13, 235, 1966. I 1 Huang, J.S., Downes, G.L. & Belzer, F.O. Utilization of fatty acids in perfused hypothermic dog kidney. f.Lipid Res. 12, 622, 1971. I2 Jellum, E., Stokke, 0. & Eldjarn, L. Screening for metabolic disorders using gas-liquid chromatogrdphy, mass spectrometry and computer techniques. Scand. J . clin. Lab. Inwst. 27, 273, 1971. 13 Kessler, G. & Lederer, H. Fluorornetric measurement of triglycerides. p. 341 in L.T. Skeggs J r (ed.) Automation in Analytical Chemistry. Terhnicon Symp. 1965. Mediad inc. New York, N.Y., 1966. 14 Lands, W.E.M. & Merkl, J. Metabolism of glycerolipids. 111. Reactivity of various acyl esters of coenzyme A with a'-acylglycerophosphorylcholine,and
15
16 17
18
19
20
21 22
23
24
25
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positional specificities in lecithin synthesis. J . b i d . Chem. 238, 898, 1963. Lerner, E., Shug, A.L., Elson, C. & Shrago, E. Reversible inhibition of adenine nucleotide translocation by long-chain fatty acyl coenzyme A esters in liver mitochondria of diabetic and hibernating animals. f. b i d . Chem. 247, 1513, 1972. Levine, J.B. & Zak, B. Automated determination of serum total cholesterol. Ctin. chim. Acta 10, 381, 1964. Linscheer, W.G., Slone, D. & Chalmers, T.C. Effects of octanoic acid on serum-levels of free fatty acids, insulin, and glucose in patients with cirrhosis and in healthy volunteers. Lancet i, 593, 1967. Mavis, R.D. & Vagels, P.R. The effect of phospholipid fatty acid composition on membranous enzymes in Escherichia coli. J. biol. Chent. 247, 652, 1972. Nesbakken, R. Aspects of free fatty acid metabolism during induced hypotherrnia. Scand. J. clin. Lab. Invest. 31, Suppl. 131, 1973. Pettersson, S., Claes, G . & Schersten, T. Fatty acid and glucose utilization during continuous hypothermic perfusion of dog kidney. Eur. surg. Res. 6 , 79, 1974. Sinensky, M. Temperature control of phospholipid biosynthesis in Escherichia culi. J . Bacteriol. 106, 449, 1971. Slaattelid, O., Flatmark, A. & Skrede, S. The importance of perfusate content of free fatty acids for kidney preservation. Scand. f. d i n . Lab. Increst. 36, 239, 1976. Trout, D.L., Estes, E.H., Jr. & Friedberg, S.J. Titration of free fatty acids of plasma: a study of current methods and a new modification. f. Lipid Rcs. 1, 199, 1960. Van Deenen, L.L.M. Phospholipids and biomembranes. p. 104 in Holman, R.T. (ed.) Progress of the chemistry of fats and other lipids. Vol 8. Pergamon Press. Oxford, 1966. Zilversrnit, D.B. Phospholipids in plasma. Stand. Meth. clin. Chem. 2, 132, 1958.
Received 17 November 1977 Accepted 15 May 1979
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney S. Skrede & O. Slaattelid To cite this article: S. Skrede & O. Slaattelid (1979) Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney, Scandinavian Journal of Clinical and Laboratory Investigation, 39:8, 765-771, DOI: 10.1080/00365517909108169 To link to this article: http://dx.doi.org/10.1080/00365517909108169
Published online: 14 Feb 2011.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [University of California Santa Barbara]
Date: 16 June 2016, At: 11:38
Scand. J. clin. Lab. Invest. 39, 765-71 1, 1979.
Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney
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S . S K R E D E & 0. S L A A T T E L I D Institute for Surgical Research, Surgical Department B and Institute of Clinical Biochemistry, Rikshospitalet, National Hospital of Norway, University Hospital, Oslo
Skrede, S. & Slaattelid, 0. Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney. Scand. J. clin. Lab. Invest. 39, 765-771, 1979. Perfusion of isolated dog kidneys was performed at 8-12°C using an albumin solution containing caprylic acid (about 6 mmol/l) and long-chain fatty acids (about 0.5 mmol/l). During 48 h the amounts of caprylic acid in the perfusate fell by about 3 mmol, whereas long-chain FFA increased by 0.2-0.3 mmol and small amounts of arachidonic acid appeared. [I4C]palmitate or [ 14C]linoleate-when added-decreased by about 10% in the perfusate. The decrease was mainly due to exchange with kidney phospholipid fatty acids. Only about 0.4% was recovered as [14C]C02.The amounts of total phospholipids in kidney tissue decreased by up to 10% during the perfusion (when 10% kidney weight gain was taken into account), whereas lysophospholipids, cholesterol, triglycerides and free fatty acids remained essentially unchanged. The distribution of fatty acids in the total phospholipid fraction was strikingly altered. The relative amounts of arachidonic acid increased, whereas all other major fatty acids decreased. Synthesis of arachidonic acid by chain elongation of perfusate [14C]linoleicacid could not be demonstrated, and no evidence was found for a significant increase of the total amounts of arachidonic acid. The possibility that arachidonic acid-containing phospholipids in the kidney are preferentially preserved during the hypothermic conditions is discussed. Key-words: arachidonic acid; fatty acid metabolism; hypothermic perfusion; kidney preservation; phospholipid fatty acids S . Skrede, M.D., Institute of Clinical Biochemistry, Rikshospitalet, Pilestredet 32, Oslo 1
In a previous study [22] we have found that the functional capacity of dog kidneys, preserved for 48 h by hypothermic perfusion, was better when free fatty acids (FFA) were present in the albumin perfusate. The concentration of total FFA in the perfusate decreased during perfusion. 0036-551 3/79/1200-0765902.00
0 1979 Medisinsk Fysiologisk Forenings Forlag
The disappearance of radiolabelled palmitic acid was rather low, however, and the results suggested that other fatty acids, e.g. caprylic acid, were removed at a higher rate than palmitic acid. In the present study, the fate of individual perfusate fatty acids during kidney perfusion has been investigated. The amounts of renal tissue lipids and the phospholipid fatty acid 765
166
S. Skrede & 0. Slaattelid
pattern before and after perfusion were also studied. MATERIAL A N D METHODS
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Reagents. The basis for the perfusion fluid was a 20% human serum albumin solution, obtained from KABI AB, Stockholm, Sweden. [I4C(U)]palmitic acid and [ 14C(U)]linoleic acid were purchased from New England Nuclear, Boston, Mass. USA. All other reagents were commercial products of high purity. Atiimnls and surgical procedures. Six mongrel dogs ( I S 1 8 kg) (exp. a-f) were used in this study. They were fasted overnight before the operative procedure, but had free access to water. The dogs underwent bilateral nephrectomy under pentobarbitone anaesthesia (Nembutal veterinary, Abott Laboratories Ltd, Chicago, Ill., USA). The dose was 30 mg/kg body weight. The right kidney was removed first and served as a control. After initial flushing [22] the kidney was weighed and placed in icecold isotonic saline. The kidney was decapsulated and we took two to four wedge-shaped biopsies (dissected free of fat); we intended to obtain mainly cortical tissue (the amount of medullary tissue was probably always less than 5 04). After weighing, the biopsies were frozen. The left kidney was then dissected free, the vessels were clamped and cut and the kidney removed. Kidney preservation and perjusiotz niedia. Immediately after removal, the left kidney was flushed, weighed and then perfused for 48 h in the Gambro perfusion machine (Gambro, Lund, Sweden) as previously described [22]. The machine was primed with a volume of 850 ml (in experiments b, e and f : 600 ml). The perfusate consisted of fatty acid-containing albumin with additives [22]. The perfusate did not contain triglycerides or cholesterol. In two experiments ['4C]palmitic acid or [14C]linoleic acid, (50 pCi) complexed with serum albumin [19] was added to the perfusate. In these experiments a C02-trap with 30% KOH was connected to the outlet of the oxygenator. After preservation, the kidney was weighed and then treated in the same manner as the right (control) kidney. During perfusion for 48 h a
gain in kidney weight was always present, and averaged 10%. Perfusate samplitig. Before perfusion started and then with intervals, perfusate samples were taken for determination of free fatty acids. Perfusate radioactivity was measured before and after kidney perfusion. When calculating the recovery of 14C-labelled fatty acids, the amounts recovered by the flush-out were also included. Samples of the perfusate were taken for sterile control. No bacterial contamination was found. Biochemical methods. 'Total free fatty acids' (mainly long-chain fatty acids) were estimated using a titrimetric method introduced by Dole [7] and modified by Trout et al. [23]. The total amounts and the relative distribution of longchain fatty acids were also determined by gas liquid chromatography (GLC) [8] (esterified fatty acids were not detected in the perfusate). Folch-extracts [9] were prepared in duplicate, and long-chain fatty acids were estimated after methylation with pentadecanoic acid as internal standard, using an 8 % BDS-column at 180°C. Caprylic acid was determined after deproteinization with methanol, acidification, extraction with diethylether and methylation, with nonanoic (pelargonic) acid as internal standard [ 121. A n 82, BDS-column was used at 110°C. The coefficients of variation for the quantitation by GLC of the individual long-chain fatty acids as calculated from nineteen duplicate perfusate analyses (including the extraction step) were: 16:O 3.4%; 61:l 9.3%; 18:O 11.3%; 18:l 4.5%; 18:2 4.4% and 20:4 (nine duplicates, after perfusion only) 7.4%. The total lipids in the perfusate as well as in the renal tissue were extracted according to Folch [9]. The recovery of [14C]palmitate from the perfusate was about 95%. In aliquots of the renal tissue lipid extract, cholesterol was estimated according to Levine & Zak [16], and triglycerides according to Kessler & Lederer [13]. Phospholipids were estimated according to Zilversmit [25]. Total phosphorous was estimated by the same method, but starting with the combustion step. Thin-layer chromatographic separation of phospholipids and cholesterol was achieved using silica gel H with a solvent mixture of petrol ether (b.p. 4O-6O0C), diethylether: acetic acid (85:15:5, v/v) [lo]. For the separation of triglycerides and free fatty acids,
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Kidney lipids during hypothermic perfusion the solvent system was hexane: diethyl-ether: acetic acid (80:20: 1, v/v). When estimating 14C-activity, the zones were visualized in an iodine vapour chamber, scraped into glass scinter funnels and repeatedly eluted using chloroform/methanol (2: 1). The phospholipid zone was in addition eluted with methanol. I4C-activity was estimated by liquid-scintillation. For quantitative lipid analyses, only standards on the lateral areas of the thin-layer plates were sprayed with 0.20,/,fluorescein. Lysolecithin and lecithin were separated by thin-layer chromatography on silica gel H, using a solvent mixture of chloroform: methano1:water:acetic acid (65: 35:5:4 v/v). Egg lecithin and lysolecithin were used as standards. The fractions were localized by spraying with 0.2% fluorescein in ethanol and eluted according to Arvidson [I]. Phospholipids were then combusted, and inorganic phosphate estimated according to Bartlett [2]. The total phospholipids were hydrolysed at 80°C for 40 min. with ethanol/KOH (30%), and the content of long-chain fatty acids was estimated as for perfusate fatty acids (see above). Caprylic acid was not detected in this fraction. A combined GLC/MC instrumentVarian MAT 112 (Varian MAT, Bremen, Western Germany) was used to confirm the structure of the peaks (also 20:4). In the experiment when [ ‘T]linoleic acid was present in the perfusate, radio-gas-chromatography of the phospholipid fatty acids were performed with a Hewlett-Packard instrument using a SB 2340 column at 200°C.
RESULTS Perfusate analyses Table I shows the results of Dole titration (‘total fatty acids’, [7, 231) at different intervals during kidney perfusion, and also the concentration of individual fatty acids as estimated by GLC. In accordance with previous results [22], a gradual decrease of the ‘Dole titre’ was found. By GLC analyses, a high concentration of caprylic acid was found in the perfusate (this medium chain fatty acid is added to the albumin solution). Table I also shows that the total fatty acids in the perfusate are underestimated by Dole’s method [7, 231, since caprylic acid is estimated with a recovery of only 50-60% by this method (own results, which agree with those of Linscheer et al. [17]). Table I shows that the decrease of caprylic acid during the perfusion is of such an extent that it explains that the ‘Dole titre’ decreases, even though the longchain fatty acids in fact increase. GLC analyses showed that the conccntration of all long-chain fatty acids increased by on an average 60% during 48 h in four experiments, and also that small amounts of arachidonic acid appear in the perfusate in this period. The total phosphorous content of the perfusate increased by about 0.4 mmol during 48 h (exp. e, results not shown). Figure 1 shows the time-course of the increase of the different long-chain fatty acids during the first 8 h of perfusion. Arachidonic acid appeared rather rapidly (after 2 h), and the increase of the
TABLE I . Changes of the fatty acid pattern of the perfusate during kidney perfusion at 10°C for 48 h . (All analyses were performed in duplicate, including the extraction step-cf. Methods.) Perfusion ‘Dole time titre’* Experiment (h) (pmol/l) a b
0
C
d C
d a b
24 48
C
d
* Cf.ref. (23), ** N.D.:
Perfusate concentration (pmol/l) 8:O
16:O
16:l
18:O
18:l
18:2
20:4
2860 3690 2740 2980
5790 6630
30
-
28 31 27 33 34 40 46
50 67 57 44 74 39
188 174 155 144 198 216 283 286 22t 283
103 126 96 88 100
N.D.** N.D. N.D. N.D.
-
181 204 156 144 197 216 272 400 218 273
100
-
129 184 107 116
27 53 17 16
2343 2040 2870 2200 1880
-*** -
2230 3350
-
~
Not detectable,
767
*** -:
36
43
100
127 70 84
Not performed
~
168
S. Skrede & 0.Slaattelid
Tissue analyses
P
T
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C 0 0
Y
U 0
x c +
8
4 Time i h )
FIG.1 . Increase of long-chain fatty acids in the perfusate during the first 8 h of kidney perfusion (exp. e). Lipid extractions according to Folch e t a / . [9] were performed in duplicate, and from each lipid extract the analyses (cf. Methods) also proceeded as duplicates. The vertical bars indicate the ‘overall SD’ for these four measurements. The symbols for the different fatty acids are: I! 16:O; 16:l; 1 8 : O ; 0 1 8 : l : 0 18:2; A 20:4
other fatty acids (e.g. 16:O) was almost linear during the first 8 h. A continued gradual increase in the level of different fatty acids is shown by Table 1. In the experiments when [‘4C]palmitic acid or [14C]Iinoleicacid was added to the perfusate, a reduction by 10% of perfusate 14C-activity was recorded during 48 h of kidney perfusion (Table 11). This I4C-activity was almost completely recovered from lipid extracts of the perfused kidney. Only traces of I4C-activity (0.36% of total radioactivity) was recovered from trapped CO,.
The amounts of phospholipids (and lysophospholipids), total cholesterol, triglycerides and free fatty acids in control and perfused kidney tissues are shown by Table 111. When it is born in mind that the wet weight of the kidney increased about loo/,, it is seen that the total phospholipids decreased by up to 10%. The lysolecithin fraction was not increased. Total cholesterol was not significantly changed during 48 h perfusion, when the precision of the method and the kidney weight increase is taken into account. The amounts of triglycerides (with the exception of experiment b) and free fatty acids were small, and were not significantly changed (even a complete wash-out of the kidney free fatty acid pool would have contributed to a minor part of the observed increase of fatty acids in the perfusate). Table IT shows that I”C-activity from palmitic acid or linoleic acid was incorporated into tissue lipids. The incorporation into the phospholipids was at a considerably higher level (more than 6% of total initial perfusate activity) than in the other lipid fractions. Before perfusion, the amounts of kidney phospholipid fatty acids was estimated to be 40-50 pmol/g wet weight. After 48 h perfusion, the amount per g wet weight decreased by on an average 15-20% (when no corrections for about 10% wet weight increase were performed). Table IV shows that the relative distribution of different phospholipid fatty acids had changed in this period. Arachidonic acid underwent a relative increase at the expense of most of the other individual fatty acids. We found no evidence for an increase of the total amounts of this fatty acid, when the precision of the methods for kidney wet weight determination and total phospholipids were taken into account. The
TABLE I I . Recovery of “C-activity after 48-h kidney perfusion with [“Clpalmitate (a) or [‘4C]linoleate (b) i n the perfusate ‘‘C-activity present in kidney lipids (% of initial [14C]palrnitate or [“C]linoleate activity in the perfusate) Time of perfusion Exp. (h) a b
48 48
Perfusate 90.9 88.5
Total extract Free C02-trap of tissue lipids Phospholipids cholesterol Triglycerides FFA 0.36 ~
8.6 12.5
6.2 6.4
0.3 0.3
0.1 0.7
1.5 5.1
Kidney lipids during hypothermic perfusion
769
TABLE 111. Pattern of the major lipid classes in the renal tissue before and after 48-h kidney perfusion
Time of perfusion Experiment (h) a
0 48 0 48 0 48
b e
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Amounts of tissue lipid (,umol/g wet weight) Kidney weight Phospholipids Total Free fatty acids (9) Total Lysolecithin cholesterol Triglycerides (sum of long-chain
* Two control
81.5 87.8 56.5 58.1 69.0 76.5
32.0 25.2 26.3 22.8 27.8 21.9
0.25 0.25 -
__ -
8.0* 7.9* 8.6 8.9
0.7 0.6 5.0 5.5 0.4 0.4
-)
I .o 0.9
kidney samples and two perfused kidney samples were analysed in quadruplicates (CV =
2%).
TABLEI V . Changes of the fatty acid pattern i n renal phsopholipids during 48-h kidney perfusion Timeof perfusion Experiment (h) a
b e f a b
0
48
No. of tissue samples
Percental distribution of major fatty acids*
2
8:O N.D.**
1
N.D.
2
N.D.
I 3
N.D. N.D.
1
N.D.
16:O 18:O 18:1 18:2 20:4 30.57 14.8 22.4 14.9 17.5 (26.8-33.1 1 (14.3-15.6) (21.6-23.8) (14.2-15.4) (16.0-19.2) 28.0 13.9 19.7 16.3 22.1 26.1 14.2 20.6 17.7 20.9 28.2 14.1 18.7 18.3 20.7 28.5; 13.5 27.1 19.2 11.5 (28.1-30.1) (13.3-13.8) (19.1-19.2) ( 1 1.1-11.9) (26.2-28.4) 26.5 12.2 17.4 17.7 26.2
* Additional minor peaks (e.g. palmitoleic acid (16:1) and unidentified peaks) always contributed with less than 3-4%, and were not included in the sum. ** Not detectable. 'f Mean of duplicate analyses (range in parentheses). Mean of three single analyses (range in parentheses).
amounts of all other fatty acids decreased, however. The composition of the kidney free fattyacidpool(l6:O 40%; 18:020%; 18:120% and 20:4 8%) did not change significantly during 48 h perfusion. In accordance with the previous study on the metabolism of [ 14C]caprylate [20], significant amounts of caprylic acid were not detected in the phospholipids of the perfused kidney by GLC/ MS-methods. In the experiment with ['4C]linoleic acid (exp. b), no incorporation of radioactivity into renal phospholipid arachidonic acid could be demonstrated by radio-gas-chromatography. (The sensitivity of the system would allow detection of a total kidney synthesis of as little as 0.3 pmol of 20:4 during 48 h.)
DISCUSSION In a previous study [22] we found that fatty acids bound to the perfusate albumin were important for the maintenance of kidney function during the preservation period. This widely used albumin-based perfusate contains caprylic acid at a high concentration (about 5-6 mmol/l) and the same long-chain fatty acids as in normal serum in about the same concentrations (sum about 0.5-0.6 mmol/l). In agreement with previous results [20], the present study shows that caprylic acid in the perfusate is rapidly utilized (by oxidation) by the dog kidney during hypothermic perfusion. Palmitic acid, on the other hand, was oxidized to COz at a very low rate.
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110
S. Skrede & 0.Sluattclid
As observed previously [ l l , 201, long-chain fatty acids (e.g. [14C]palmiticor fl"C]linoleate) were incorporated into tissue phospholipids, and to a lower extent into kidney triglycerides. In the free cholesterol fraction, traces of I4C from palmitic acid of linoleic acid were recovered, probably by de iiow synthesis of cholesterol utilizing acetate generated by breakdown of the fatty acid. Kidney total phospholipids decreased by up to lox, without an increase in the lysophospholipids. Concomitantly, long-chain fatty acids (and total phosphorous) in the perfusate increased, and arachidonic acid also appeared. Previously Pettersson et a/. [20] did not find this decrease of the tissue phospholipids using essentially the same conditions for perfusion as we did, whereas Huang et al. [I 11 found that phospholipids were lost during kidney perfusion with cryoprecipitated plasma perfusate. Our finding that long-chain fatty acids in the perfusate increased during the perfusion is at variance with the report of Pettersson et al. [20], who also stated very high concentrations of individual fatty acids before perfusion (e.g. 18:l at almost 2000 pmol/l). The analytical performance of the quantitative gas chromatographic analyses of long-chain fatty acids in the present study has been documented (cf. Material and methods). The decrease of kidney phospholipids evidently reflects a changed balance between catabolism and synthesis. Possibly, de nouo phospholipid synthesis becomes reduced because of a loss of cytidine nucleotides, although Collste [6] did not find great changes of the total adenine nucleotide pool during kidney perfusion. I t is well known that when the temperature is lowered, e.g. in hibernating mammals [15] as well as in lower organisms [18], the phospholipids will contain a greater amount of unsaturated fatty acids. Changes in the pattern of long-chain fatty acids in the phospholipid fraction during hypothermic kidney perfusion have not been reported previously. We found a fatty acid pattern prior to perfusion which was in accordance with previous studies [5]. During 48 h perfusion, however, the relative distribution of fatty acids changed remarkably, with a great relative increase of arachidonic acid. The kidney has efficient fatty acid chain elonga-
tion mechanisms [3], but we did not find 14Cincorporation into arachidonic acid from perfusate [14C]linoleic acid. The cause of the changed distribution was that no significant changes in arachidonic acid were present, while all other fatty acids decreased. Arachidonic acid and other unsaturated fatty acids are predominantly found in the 2position of the phospholipids, which is more 'metabolically active' than the 1-position (241. The fatty acid in the 2-position is subjected to a cycle of deacylation (by phospholipase A l ) and a reacylation (acyl-CoA: lysolecithin acyl-transferase [14]. The present study does not give evidence to show whether a slow deacylation of arachidonylic species occurred, or whether a rapid reacylation of this fatty acid took place. In favour of the latter mechanism is that acylCoA : lysolecithin acyl-transferase preferentially reincorporates unsaturated fatty acids [ 141, and that acyl-CoA hydrolase is more active with short-chain unsaturated fatty acids [14]. The chainlength of the fatty acid may also be of importance for the reincorporation, since arachidonic acid is incorporated more rapidly than oleic acid by this mechanism [4]. In relation to our observations, it is interesting that in E. coli, the acyl-transferase is relatively more active with the unsaturated fatty acids than with the saturated ones when the temperature is lowered [l8, 211. This may explain the greater content of unsaturated fatty acids in the phospholipid fraction of bacteria at low temperatures. A similar temperaturedependence may be the reason for the preservation of arachidonic acid in the phospholipid fraction of the mammal kidney during hypothermic preservation. Other mechanisms are also possible, e.g. that phospholipase A l is relatively less active at lower temperatures against phospholipids containing arachidonic acid. ACKNOWLEDGMENT The excellent technical assistance of Ms Anne Marie Lund is gratefully acknowledged. REFERENCES 1 Arvidson, G.A.E. Structural and metabolic hetero-
genity of rat liver glycerophosphatides. Eur. J. Biochem. 4, 478, 1968.
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Kidney lipids during hypothermic perfusion 2 Bartlett, G.R. Phosphorus assay in column chromatography. J . biol. Chem. 234, 466, 1959. 3 Bojesen, I., Bojesen, E. & Capito, K. In vitro and in vivo synthesis of long-chain fatty acids from (I-'4C)acetate in the renal papillae of rats. Biochint. brophys. Acta 424, 8, 1976. 4 Brandt, A.E. & Lands, W.E.M. The effect of acylgroup composition on the rate of acyl transferasecatalyzed synthesis of lecithin. Biuchini. biopltys. Acta 144, 605, 1967. 5 Butkus, A., Robertson Lazzarini, A., Ehrhart, L.A. & Lewis, L. Renal lipids of dogs fed on arteriosclerosis-inducing diet. Exp. molec. Path. 17, 55, 1972. 6 Collste, H. Preservation of kidneys for transplantation. Experimental studies. Acta chir. scanrl. Suppl. 425, 1972. 7 Dole, V.P. A relation between non-esterified fatty acids in plasma and the metabolism of glucose. J . clin. Inivst. 35, 150, 1956. 8 Eldjarn, L., Try, K . , Stokke, O., Munthe-Kaas, A.W., Refsum, S., Steinberg, D., Avigan, J. & Mire, C. Dietary effects on serum-phytanic-acid levels and on clinical manifestations in heredopathia atactica polyneuritiformis. Lancet i, 691, 1966. 9 Folch, J., Lees, M. & Sloane Stanley, G.H. A simple method for the isolation and purification of total lipids from animal tissues. f. b i d . Chem. 226, 497, 1957. 10 Gloster, J . & Fletcher, R.F. Quantitative analysis of serum lipids with thin-layer chromatography. Clin. chim. Acta 13, 235, 1966. I 1 Huang, J.S., Downes, G.L. & Belzer, F.O. Utilization of fatty acids in perfused hypothermic dog kidney. f.Lipid Res. 12, 622, 1971. I2 Jellum, E., Stokke, 0. & Eldjarn, L. Screening for metabolic disorders using gas-liquid chromatogrdphy, mass spectrometry and computer techniques. Scand. J . clin. Lab. Inwst. 27, 273, 1971. 13 Kessler, G. & Lederer, H. Fluorornetric measurement of triglycerides. p. 341 in L.T. Skeggs J r (ed.) Automation in Analytical Chemistry. Terhnicon Symp. 1965. Mediad inc. New York, N.Y., 1966. 14 Lands, W.E.M. & Merkl, J. Metabolism of glycerolipids. 111. Reactivity of various acyl esters of coenzyme A with a'-acylglycerophosphorylcholine,and
15
16 17
18
19
20
21 22
23
24
25
771
positional specificities in lecithin synthesis. J . b i d . Chem. 238, 898, 1963. Lerner, E., Shug, A.L., Elson, C. & Shrago, E. Reversible inhibition of adenine nucleotide translocation by long-chain fatty acyl coenzyme A esters in liver mitochondria of diabetic and hibernating animals. f. b i d . Chem. 247, 1513, 1972. Levine, J.B. & Zak, B. Automated determination of serum total cholesterol. Ctin. chim. Acta 10, 381, 1964. Linscheer, W.G., Slone, D. & Chalmers, T.C. Effects of octanoic acid on serum-levels of free fatty acids, insulin, and glucose in patients with cirrhosis and in healthy volunteers. Lancet i, 593, 1967. Mavis, R.D. & Vagels, P.R. The effect of phospholipid fatty acid composition on membranous enzymes in Escherichia coli. J. biol. Chent. 247, 652, 1972. Nesbakken, R. Aspects of free fatty acid metabolism during induced hypotherrnia. Scand. J. clin. Lab. Invest. 31, Suppl. 131, 1973. Pettersson, S., Claes, G . & Schersten, T. Fatty acid and glucose utilization during continuous hypothermic perfusion of dog kidney. Eur. surg. Res. 6 , 79, 1974. Sinensky, M. Temperature control of phospholipid biosynthesis in Escherichia culi. J . Bacteriol. 106, 449, 1971. Slaattelid, O., Flatmark, A. & Skrede, S. The importance of perfusate content of free fatty acids for kidney preservation. Scand. f. d i n . Lab. Increst. 36, 239, 1976. Trout, D.L., Estes, E.H., Jr. & Friedberg, S.J. Titration of free fatty acids of plasma: a study of current methods and a new modification. f. Lipid Rcs. 1, 199, 1960. Van Deenen, L.L.M. Phospholipids and biomembranes. p. 104 in Holman, R.T. (ed.) Progress of the chemistry of fats and other lipids. Vol 8. Pergamon Press. Oxford, 1966. Zilversrnit, D.B. Phospholipids in plasma. Stand. Meth. clin. Chem. 2, 132, 1958.
Received 17 November 1977 Accepted 15 May 1979
