PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS Prostaglandins Leukotnenes and Essenr~alFatty Aads (1992) 47. 203-207 Q Longman Group UK Ltd 1992
Effect of Heat Inactivation and Freezing on Fatty Acid Composition Plasma and Red Blood Cells
of
V. L. Brown, J. C. Shay and N. L. Morse-Fisher Efamol Research Institute, P.O. Box 818. Kentville. Nova Scotia, Canada B4N 4H8 (Reprint requests to NLM-F) ABSTRACT. The effect of heat inactivation and freezing on fatty acid composition of plasma and red blood cells was investigated. Analysis was completed at baseline; after freezing; after incubation; after incubation and subsequent freezing; after incubation, freezing and a second incubation; and after freezing and subsequent incubation. There were changes in fatty acid levels observed in all groups with the phospholipid fractions showing the greatest changes. Those bloods that had been incubated, frozen and incubated again, and those which had been frozen initially followed by incubation showed the greatest change when compared to baseline samples. Even though there were changes in fatty acid levels seen in all groups, the changes were small except in those two groups. Treatment of blood with either of those two treatment regimens changes the fatty acid values so that they do not accurately reflect the composition of fatty acids in the blood.
INTRODUCTION
ment regimens and the effect of those regimens acid composition of RBCs and plasma.
Numerous clinical trials have been designed to measure differences in blood fatty acid composition in normal individuals and in those bith various disease conditions (16). One of these studies has shown abnormal fatty acid levels in acquired immune deficiency syndrome (AIDS) patients (7). Risk of contamination is becoming an increasingly serious consideration for health professionals working with human blood samples, particularly those samples known to contain HIV. When clinical trial blood samples are received, it is often difficult to determine if the samples have been screened for such infectious diseases as AIDS or hepatitis B. It has been reported (8-14) that heat inactivation of plasma and red blood cells (RBCs) at 55- 60 “C for 3 h will completely destroy the infectivity of the retroviruses responsible for these and other diseases. Many laboratories are therefore routinely using this procedure. The effect of heat inactivation on the fatty acid content of red blood cells and plasma has not been determined; the results presented here will illustrate that effects. Blood samples from clinical trials are frozen for transit, on occasion thaw during shipment and are often refrozen upon receipt. They may also be heat inactivated, frozen for transit and inactivated a second time for safety reasons. In this study we have therefore considered a variety of possible treat-
MATERIALS
on fatty
AND METHODS
In the initial study, a combined total of 150 mL of whole blood was collected from 8 healthy volunteers. The samples were maintained on ice for not more than 30 min until all were available for simultaneous centrifugation to separate RBCs and plasma. The RBCs from all patients were then pooled as was the plasma. An initial 12 x 1 mL aliquots of plasma and RBCs were removed from the pools and analysed immediately. These samples served as baseline samples. 15 mL of RBCs and plasma were immediately frozen at -20 “C for 1 week, after which 12 x 1 mL aliquots were analysed for fatty acid content. The remaining blood samples were incubated in a water bath at 55 “C for 3 h. 12 x 1 mL samples were then removed and analysed for fatty acid content, and the remaining samples frozen at -20 “C for 1 week. 12 x 1 mL samples were removed from the pool and analysed immediately upon thawing. The remainder were again incubated at 55 “C for 3 h and analysed (Fig.). Table 1 explains the group numbers seen in Tables 2 and 3. A second study was completed consisting of baseline samples, and samples frozen for 1 week followed by a 3-h incubation at 55 “C. Blood collection and preparation in this second experiment were similar to that described previously.
Date received 23 March 1992 Date accepted 1.5May 1992 203
204
Prostaglandins Leukotrienes and Essential Fatty Acids
Table 1
Blood
Group numbers
Group FA
Description
analysis Day
I
1
Incubation
2 3
FR
for treatment regimens (initial study)
analysis
Day 7
I
4
Incubation
5
Figure Flow diagram illustrating the procedure used in the analysis of RBCs and plasma (initial study).
All plasma and RBC samples were extracted by the method of Folch et al (15). Total phospholipids from RBCs; and plasma total phospholipids, triglycerides and cholesterol esters were separated by thin layer chromatography. Fatty acid compositions were assayed by gas chromatography as described previously with the following changes; the column packing used was GP 10% SP-2330 on 100/l 20 Chromosorb WAW (Supelco Canada Ltd, Oakville, Ontario) and the final temperature was 195 “C ( 16). Briefly, the lipid bands were removed from the thin layer chromatography plates, methylated using fresh (daily) boron trifluoride in methanol (Supelco Canada Ltd, Oakville, Ontario) at 90 “C for
Baseline Samples: Fatty acid results obtained from analysis of RBCs and plasma immediately upon separation. Fatty acid results obtained from the analysis of RBCs and plasma after initial 3-h incubation at 55 “C. Fatty acid results obtained from the analysis of RBCs and plasma after I week at -20 “C. No initial incubation. Fatty acid results obtained from the analysis of RBCs and plasma after initial 3-h incubation at 55 “C and subsequent freezing for I week at -20 “C’. Fatty acid results obtained from the analysis 01 RBCs and plasma after initial 3-h incubation at 55 “C. subsequent freezing for I week at -20 “C. and a second 3-h incubation at 55 “C.
30 min and analysed on the same 6 ft glass gas chromatography column. Student’s t-test was used to calculate the significance of differences among the groups.
RESULTS The detailed results of fatty acid composition of RBC total phospholipids; and plasma total phospholipids, triglycerides. and cholesterol esters from the initial study are shown in Tables 2 and 3. There are changes in fatty acid levels evident in all blood fractions of all groups, especially in the phospholipid fractions. Those blood
Table 2 Total phospholipid composition of RBCs and plasma before and after freezing and heat inactivation (initial study) (results expressed as area percent) Group
I
Group 2
Plusma torol phospholipids 16:O 25.94 f 0.51 18:O 12.69 f I .05 l&In9 15.32 f 2.02 I Wnh 22.52 f 0.55 l8:3n6 0.02 f 0.06 I8:3n3 0.25 f 0.06 20:3n6 3.01 f 0.29 20:4n6 I I .34 + 0.90 20:5n3 0.72 ? 0.06 22:4n6 0.56 + 0.07 22:5n6 0.68 + 0.14 22:Sn3 0.84 ?r 0.08 22:6n3 2.91 -I 0.28
27.30 f 12.30 f 14.43 f 22.20 f 0.00 * 0. I8 & 3.26 ? I I .x0 f 0.69 k 0.40 * 0.33 * 0.90 t 3. IS *
RBC /oral phospholipids l6:O 20.3 I k 0.26 IS.72 * 0.5 I l8:O i&In9 17.03 * 0.48 18:2n6 10.98 f 0.20 IX:3nh 0.00 f 0.00 I8:3n3 0.18 f 0.02 I.71 f 0.02 20:3nh 20:4nh 16.76 f 0.21 20:5n3 0.62 f 0.03 22:4nh 3.65 f 0. I3 22:5n6 1.61 f0.17 22:5n3 2.24 + 0.08 22:6n3 4.01 f 0.08
20.60 + 0.5X 15.85 f I .02 18. IX f 0.36** I I .68 k 0.29** 0.00 * 0.00 0.01 I!zo.O4** I .58 + 0.03** 17.63 + 0.44** 0.59 * 0.03* 3.40 f 0.07** 0.99 * 0. IO** 2.43 + 0.06** 4.36 + 0. I2**
0.37** 0.66 0.15 0.38 0.00 0.02** 0. IO* 0.20 0.02 0.04** 0.06** 0.02*
0.04*
Group 3
Group 4
Group 5
27.68 ? 0.47** 13.37 * 0.24* 14.9s * 0.24 22.9 I * 0.52 0.0I * 0.05 0.14 + 0.07** 2.76 f O.O7* Il.l6f0.12 0.6X f 0.0 I 0.42 f 0.05** 0.42 f O.lh** 0.79 I? 0.03 2.82 f 0.05
27.59 +0.57** 13.6 I + 0.29* 14.77 & 0.28 2 I .S I ? 0.36** o.OO & 0.00 0. I Y * 0.03* 2.99 IL 0.14 I I.2.5 + 0.30 0.66 * 0.03** 0.46 * 0.08** 0.50 f 0.14** 0.83 + 0.04 2.99 f 0. I2
1x.54*2.11** IS.68 i 0.45** IS.74 * 0.25 1-J 31 + 0.64 --.. 0.00 ?zMM) 0. I9 * o.oh* 3.‘) I + 0.32** 13.77 * 0.73** 0.x.3 & o.o’)** OS8 ?r 0. IO 0.63 + 0. I2 I .03 + 0.06** 3.84 * 0.2.5**
2 I .6S 15.44 17.2 I I I .3s 0.00 0.12 I .62 16.28 0.59 3.38 I .48 2.08 3.78
20.64 16.20 17.X9 I I .40 0.00 0. I I I .6S 17.04 0.59 3.50 I .3Y 2.1’) 4.0 I
17.62 13.43 17.54 12.56 (I.00 0.2 I I .Y7 IV.86 0.90 3.73 I .hl 2.x4 5. I6
The results are expressed as mean + SD. * Significantly different from the baseline group al p < 0.05 ** Significantly different from the baseline group at p < 0.01
+ + f * * * ? + * * * * f
0.39** 0.56 0.2x 0.20** 0.00 0.07* 0. IO* 0.39** 0.04 0.17** 0.25 0.08** 0.16**
* + f f f + f * * + + * i
0.7 I 0.80 0.61** 0.34** 0.00 0.07** 0. I2 0.3 I * 0.05 0.09** 0.10** 0.06 0. IO
f + + + f * f & * * ? * +
I .02** I .43** 0.43* 0.66** o.oft 0.05 0.1 o** 0.93** 0.2 I * 0.3 I 0.35 0.09** 0.X**
Effect of Heat Inactivation
and Freezing on Fatty Acid Composition
Table 3 Plasma triglyceride and cholesterol ester composition (initial study) (results expressed as area percent)
Group 1 Plasma triglwerides 16:O 22.88 * 0.38 18:O 3.61 k 0.17
18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3 Plasnta 16:0 18:O
18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3
44.01 * 1.01 17.19 i 1.41 0.34 * 0.19 0.98 k 0.05 0.18kO.11 1.18 f 0.20 0.00 + 0.00 0.00 f 0.00 0.00 f 0.00 0.21 i 0.1 I 0.27 f 0.14 cholesterol
23.20 f 0.18* 3.22 f 0.25** 43.12 f 0.49* 17.25kO.46 0.40 f 0.09
1.OOf 0.06 0.17 f 0.02 1.08 + 0.04 0.00 f 0.00 0.00 + 0.00 0.00 f 0.00 0.14f0.13 0.31 * 0.10
23.48 3.86 44.71 16.90 0.21 0.82 0.15 1.13 0.03 0.00 0.00 0.00 0.14
205
before and after freezing and heat inactivation
Group 3
Group 2
of Plasma and Red Blood Cells
f + * f * + + * + * -t i *
0.3 I** 0.12** 1.11 0.52 0.15 0.26 0.07 0.10 0.07 0.00 0.00 o.oo** 0.17
Group 4
Group 5
23.50 3.67 44.04 16.65 0.33 0.89 0.15 I .06 0.00 0.00 0.00 0.00 0.16
* 0.40** k 0.07 k 0.27 + 0.15 f 0.08 + 0.07** f 0.05 f 0.04 f 0.00 rt 0.00 a? 0.00 + o.oo** f 0.16
14.26 4.98 52.92 19.78 0.18 1.08 0.15 1.54 0.00 0.00 0.00 0.03 0.08
f i. k i f f f + f f + k f
2.93** 0.47** 3.41** 1.37** 0.35 0.06** 0.10 0.18** 0.00 0.00 0.00 0.09** 0.17**
11.23 1.20 20.51 50.99 0.95 0.36 0.64 7.87 0.68 0.00 0.00 0.00 0.72
k 5 k k r f + f f f + + +
6.02 1.82 21.28 52.88 0.79 0.47 0.90 10.59 0.86 0.01 0.00 0.00 I .20
+ k f k i * k * f f + + f
2.97** 0.39** 0.97** 1.55* 0.08** 0.13 0.15** 2.28** 0.29 0.04 0.00 0.00 0.76**
esters
10.97 f 0.48 1.01 kO.11 20.18 k 0.53 5 I .37 + 0.77 1.10~0.12 0.44 + 0.06 0.65 + 0.02 7.61 k 0.20 0.70 k 0.05 0.00 k 0.00 0.00 f 0.00 0.00 + 0.00 0.48 f 0.02
10.32 1.01 19.80 51.59 1.13 0.48 0.73 8.03 0.71 0.00 0.00 0.00 0.52
f 0.81* f 0.09 f 1.10 f 1.14 kO.08 + 0.06 f 0.07** + 0.63 f 0.06 f 0.00 f 0.00 + 0.00 k 0.18
11.48f 0.75 1.41 f 0.77 20.36 + 0.91 50.41 k 2.36 0.90 i 0. IO** 0.36fO.11 0.62 k 0.02** 7.83 + 0.28 0.68 f 0.05 0.02 f 0.07 0.02 + 0.06 0.00 +_0.00 0.83 f 0.42*
0.45 0.38 0.31
1.04 0.14* 0.06* 0.03 0.22* 0.05 0.00 0.00 0.00 0.20**
The results are expressed as mean + SD. * Significantly different from the baseline group at p < 0.05 ** Significantly different from the baseline group at p < 0.01
samples which were incubated at 55 “C, frozen for 1 week and reheated at 55 “C (group 5) showed the greatest significant difference compared to baseline. The percentages of samples per group differing from baseline by less than 5%, between 5.1-9.9%, between lO-19.9% or greater than 20% were calculated from the fatty acid results. While groups 2-4 showed significant differences, few showed changes greater than 10% in any fatty acid when compared to corresponding baseline samples. Group 5 showed differences greater than 10% and often greater than 40%. The results of the second study are shown in Tables 4 and 5. As seen in Table 4, there are some significant changes in fatty acid levels in plasma and RBC phospholipids when compared to baseline. As seen in Table 5, there are also significant changes in plasma triglyceride and cholesterol esters.
DISCUSSION It is now well known that treatment of blood suspected of harbouring HIV and other retroviruses with temperatures of 55-60 “C for 30 min or more will completely destroy the infectivity of the virus present (8-14). Because this procedure is common and in fact advised, and because the effect of this treatment on fatty acid levels has not been determined, we compared the fatty acid levels in RBC phospholipids; and plasma phospholipids,
triglycerides and cholesterol esters to study changes observed after various treatment regimens involving heating and freezing. In the initial study, there were changes in fatty acid levels observed in all fractions studied when compared to baseline samples. Previous work done in our laboratory has shown that plasma and RBC fatty acid values change after storage at 4 “C for as little as 2 h. In that study, after the 2-h period, RBC levels showed significant but small changes from baseline in every fatty acid examined with the exception of palmitic and oleic acids (unpublished results). The small changes (less than 10%) seen in fatty acid levels in all groups in this study may reflect this almost immediate storage effect. The fatty acids of blood samples which were incubated at 55 “C, frozen and then reheated (group 5) showed the largest change from initial values (group 1). A high percentage of these fatty acid values differed from controls by more than 20% and some by as much as 40%. The results from the second experiment showed significant changes in fatty acid patterns after freezing followed by heat inactivation when compared to baseline values. In an unpublished study, we treated plasma and RBCs immediately upon separation with an antioxidant butylated hydroxy-toluene (BHT), to ascertain if the presence of an antioxidant could decrease the degree of change in fatty acid levels after freezing and heat inactivation. Solubility of BHT in plasma and RBC proved to be a problem. Fatty acid levels of plasma and RBCs
206
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Table 4 Total phospholipid composition of RBC and plasma before and after freezing and heat inactivation (second study) (results expressed as area percent) Baseline
Freezing and incubation
Plasma total pl~ospl~olipids
16:O 18:O 18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3
25.40 11.38 14.58 24.46 0.00 0.26 3.06 12.13 0.00 0.49 0.52 0.76 3.32
? 0.61 + 1.46 * 0.73 + 1.86 z+0.00 * 0.03 F 0.35 !c 0.93 f 0.00 f 0.10 f 0.12 f 0.03 f 0.25
Table 5 Plasma triglyceride and cholesterol ester composition before and after freezing and heat inactivation (second study) (result\ expressed as area percent)
26.33 12.41 16.77 23.57 0.00 0.14 2.99 10.12 0.75 0.34 0.30 0.65 2.85
? + * * * * i f ? + * f f
0.61 0.70 0.22** 0.47 0.00 0.02** 0.15 0.35** 0.08** 0.06* 0.19** 0.06** 1.23
Baseline P/usrm~ tr-i,q/yerides 16:O
18:O 18:ln9 18:3n6 IX:3n6 18:3n3 30:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 226n.3 Phsnw
RBC rota1 p~tosp~oiipids l&O 18:0
20.58 + 0.43 12.81 k 0.29
21.42i 1.08* 15.08 k 0.63**
16:O
18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3
16.76 13.16 0.00 0.21 1.86 16.38 0.00 3.34 1.25 2.10 4.69
18.45 11.40 0.00 0.18 1.48 14.36 0.62 3.43 1.47 1.88 4.09
18:lnY 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 ?2:4n6 22:5n6 22:5n3 22:6n3
f + f * + f * * f + *
0.19 0.27 0.00 0.01 0.02 0.51 0.00 0.07 0.06 0.06 0.17
f f * * f * f + f f *
0.78** 0.53** 0.00 0.08 0.21** 1.23** 0. IO** 0.23 0.19** 0.18** 0.34**
The results are expressed as mean + SD. * Significantly different from the baseline group at p < 0.05 ** Significantly different from the baseline group at p < 0.01
analysed immediately, and after freezing and heat inactivation, were compared. It was found that the antioxidant was ineffective at preventing changes in fatty acid values. We conclude that RBCs and plasma fatty acid levels are not substantially affected by a 3-h incubation at 55 “C, or by freezing at -20 “C for a period of 1 week or even by these two treatments combined. However we do conclude that a second incubation, after the initial heating and freezing, changes the fatty acid values so that they do not accurately reflect the composition of fatty acids in the blood of patients. We also conclude that heat inactivation following an initial freezing yields unsatisfactory results. From these results, it is clear that blood samples suspected of being contaminated with the HIV virus or other retroviruses can be treated with a 3-h incubation at 55 “C before any freezing and shipment occur, without adverse effects on the fatty acid composition. References 1. Rocklin R. Manku MS, Morse N. Plasma, red blood cell and white cell phospholipid essential fatty acids in atopic subjects with respiratory symptoms. Prog Lipid Res 1986: 25: 203-204 2. Manku MS. Hoi-robin DF, Morse NL, Morse NL, Wright S, Burton JL. Essential fatty acids in the plasma phospholipids of patients with atopic eczema. Br J Dermatol 1984: 110 (6): 643-648
I8:O
cholesteroi
22.18 -t 0.62 3.43 + 0.13 43.21 f 0.36 17.17f0.16 0.30 f 0.04 1.09 f 0.05 0.1’20.09 I .08 f 0.06 0.00 * 0.00 0.00 * 0.00 0.00 * 0.00 0.00 * 0.00 0.52 * 0.02
Freezing and incubation
22.27 3.26 45.34 15.77 0.23 0.84 0.12 0.71 0.14 0.07 0.00 0.15 0.38
f f + * * + * * i: + t * +
0.68 0. IJ* 0.44** 0.86** 0.15 0.0X*” 0.05 0.03** 0.02** 0.02** 0.00 0.01** 0.0 I **
esteiJ
10.12 0.84 20.09 54.30
f f f + 1.06f 0.52 It 0.62 f 6.91 f 0.00 f 0.00 * 0.00 + 0.00 * 0.45 *
0.28 0.08 1.Oh 0.89 0.08 0.05 0.03 0.30 0.00 0.00 0.00 0.00 0.06
11.01f ()..&$“” 1.00 2 I .77 53.13 0.99 0.48 0.53 5.44 0.42 0.00 0.00 0.00 0.28
* * f * * + + * * * + +
0.13** 0.53* 1.40* 0.20 0.07 0.05** 0.4 1x* 0.12** 0.00 0.00 0.00 0. I I **
The results are expressed as mean f SD. * Significantly different from the baseline group at p < 0.05 ** Significantly different from the baseline group at p < 0.01
3. Manku MS. Horrobin DF. Huang YS. Morse N. Fatty acids in plasma and red cell membranes in normal humans. Lipids 1983: 18(12): 906908 4. Hotrobin DF, Ells K, Morse-Fisher N. Manku MS. Fatty acid distribution in plasma phospholipids in normal individuals from different geographical locations. J Nutr Med 1991: 249-258 5. Wright S. Morse N. Manku MS. Essential fatty acids in plasma of patients with leprosy. Int J Lepr 1991; 59(2): 271-277 6. Thuluvath PJ. Triger DR, Manku MS, Morse-Fisher N. Evening primrose oil in the treatment of severe refractory biliary pruritis. Eur J Gastroent Hepatol 1991: 3: X7-90 7. Begin ME, Manku MS. Horrobin DF. Plasma fatty acid levels in patients with acquired immune deficiency syndrome and in controls. Prostaglandins Leukot Essent Fatty Acids 1989; 37: 135-I 37 8. Spire B, Dormont D, Barre-Sinoussi F. Montagnier L. Chermann JC. Inactivation of lymphadenapathyassociated virus by heat, gamma rays and ultraviolet light. Lancet 1985: i: 188-189 Ginosa W. Inactivation of viruses by ionizing radiation\ and by heat. In: Maramorosch K. Koprowski H (eds). Methods in virology IV. New York: Academic Press. 1968: 139-209 McDougal JS. Martin LS. Con SP. Mozen M. Heldebrant CM, Evatt BL. Thermal inactivation of acquired immunodeficiency syndrome virus, human T lymphotropic virus-III/lymphadenopathy-associated virus. with special reference to antihemophilic factor. J Clin Invest 1985; 76: X75-877 Dolana G. Hollinger FB. Thomas W. Gyorkey F. Continued observations on the effect of a heating procedure on the inactivation of non-A, non-B hepatitis and hepatitis B viruses in clotting factor concentrate. Thromb Haemost 1983; 50: 115 Martin LS, McDougal JS. Loskoski SL. Disinfection and
Effect of Heat Inactivation inactivation of the human T lymphotropic virus type III/ lymphadenopathy-associated virus. J Infect Dis 1985: 152 (2): 4wo3 13. Klein M, Deforest A. Principles of viral inactivation. In: Block SS (ed) Disinfection, sterilization, and preservation 3rd ed. Philadelphia: Lea and Febiger 1983; 422434 14. Einarsson M, Perenius L, McDougal JS. Cort S. Heat inactivation of human immunodeficiency virus in
and Freezing on Fatty Acid Composition
of Plasma and Red Blood Cells
solutions of antithrombin III. Transfusion 1989; 229(2): 148-152 15. Folch J. Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957: 226:497 16. Manku MS, Horrobin DF, Huang YS, Morse N. Fatty acids in plasma and red cell membranes in normal humans. Lipids 1983; 18(12): 906908
207
Effect of Heat Inactivation and Freezing on Fatty Acid Composition Plasma and Red Blood Cells
of
V. L. Brown, J. C. Shay and N. L. Morse-Fisher Efamol Research Institute, P.O. Box 818. Kentville. Nova Scotia, Canada B4N 4H8 (Reprint requests to NLM-F) ABSTRACT. The effect of heat inactivation and freezing on fatty acid composition of plasma and red blood cells was investigated. Analysis was completed at baseline; after freezing; after incubation; after incubation and subsequent freezing; after incubation, freezing and a second incubation; and after freezing and subsequent incubation. There were changes in fatty acid levels observed in all groups with the phospholipid fractions showing the greatest changes. Those bloods that had been incubated, frozen and incubated again, and those which had been frozen initially followed by incubation showed the greatest change when compared to baseline samples. Even though there were changes in fatty acid levels seen in all groups, the changes were small except in those two groups. Treatment of blood with either of those two treatment regimens changes the fatty acid values so that they do not accurately reflect the composition of fatty acids in the blood.
INTRODUCTION
ment regimens and the effect of those regimens acid composition of RBCs and plasma.
Numerous clinical trials have been designed to measure differences in blood fatty acid composition in normal individuals and in those bith various disease conditions (16). One of these studies has shown abnormal fatty acid levels in acquired immune deficiency syndrome (AIDS) patients (7). Risk of contamination is becoming an increasingly serious consideration for health professionals working with human blood samples, particularly those samples known to contain HIV. When clinical trial blood samples are received, it is often difficult to determine if the samples have been screened for such infectious diseases as AIDS or hepatitis B. It has been reported (8-14) that heat inactivation of plasma and red blood cells (RBCs) at 55- 60 “C for 3 h will completely destroy the infectivity of the retroviruses responsible for these and other diseases. Many laboratories are therefore routinely using this procedure. The effect of heat inactivation on the fatty acid content of red blood cells and plasma has not been determined; the results presented here will illustrate that effects. Blood samples from clinical trials are frozen for transit, on occasion thaw during shipment and are often refrozen upon receipt. They may also be heat inactivated, frozen for transit and inactivated a second time for safety reasons. In this study we have therefore considered a variety of possible treat-
MATERIALS
on fatty
AND METHODS
In the initial study, a combined total of 150 mL of whole blood was collected from 8 healthy volunteers. The samples were maintained on ice for not more than 30 min until all were available for simultaneous centrifugation to separate RBCs and plasma. The RBCs from all patients were then pooled as was the plasma. An initial 12 x 1 mL aliquots of plasma and RBCs were removed from the pools and analysed immediately. These samples served as baseline samples. 15 mL of RBCs and plasma were immediately frozen at -20 “C for 1 week, after which 12 x 1 mL aliquots were analysed for fatty acid content. The remaining blood samples were incubated in a water bath at 55 “C for 3 h. 12 x 1 mL samples were then removed and analysed for fatty acid content, and the remaining samples frozen at -20 “C for 1 week. 12 x 1 mL samples were removed from the pool and analysed immediately upon thawing. The remainder were again incubated at 55 “C for 3 h and analysed (Fig.). Table 1 explains the group numbers seen in Tables 2 and 3. A second study was completed consisting of baseline samples, and samples frozen for 1 week followed by a 3-h incubation at 55 “C. Blood collection and preparation in this second experiment were similar to that described previously.
Date received 23 March 1992 Date accepted 1.5May 1992 203
204
Prostaglandins Leukotrienes and Essential Fatty Acids
Table 1
Blood
Group numbers
Group FA
Description
analysis Day
I
1
Incubation
2 3
FR
for treatment regimens (initial study)
analysis
Day 7
I
4
Incubation
5
Figure Flow diagram illustrating the procedure used in the analysis of RBCs and plasma (initial study).
All plasma and RBC samples were extracted by the method of Folch et al (15). Total phospholipids from RBCs; and plasma total phospholipids, triglycerides and cholesterol esters were separated by thin layer chromatography. Fatty acid compositions were assayed by gas chromatography as described previously with the following changes; the column packing used was GP 10% SP-2330 on 100/l 20 Chromosorb WAW (Supelco Canada Ltd, Oakville, Ontario) and the final temperature was 195 “C ( 16). Briefly, the lipid bands were removed from the thin layer chromatography plates, methylated using fresh (daily) boron trifluoride in methanol (Supelco Canada Ltd, Oakville, Ontario) at 90 “C for
Baseline Samples: Fatty acid results obtained from analysis of RBCs and plasma immediately upon separation. Fatty acid results obtained from the analysis of RBCs and plasma after initial 3-h incubation at 55 “C. Fatty acid results obtained from the analysis of RBCs and plasma after I week at -20 “C. No initial incubation. Fatty acid results obtained from the analysis of RBCs and plasma after initial 3-h incubation at 55 “C and subsequent freezing for I week at -20 “C’. Fatty acid results obtained from the analysis 01 RBCs and plasma after initial 3-h incubation at 55 “C. subsequent freezing for I week at -20 “C. and a second 3-h incubation at 55 “C.
30 min and analysed on the same 6 ft glass gas chromatography column. Student’s t-test was used to calculate the significance of differences among the groups.
RESULTS The detailed results of fatty acid composition of RBC total phospholipids; and plasma total phospholipids, triglycerides. and cholesterol esters from the initial study are shown in Tables 2 and 3. There are changes in fatty acid levels evident in all blood fractions of all groups, especially in the phospholipid fractions. Those blood
Table 2 Total phospholipid composition of RBCs and plasma before and after freezing and heat inactivation (initial study) (results expressed as area percent) Group
I
Group 2
Plusma torol phospholipids 16:O 25.94 f 0.51 18:O 12.69 f I .05 l&In9 15.32 f 2.02 I Wnh 22.52 f 0.55 l8:3n6 0.02 f 0.06 I8:3n3 0.25 f 0.06 20:3n6 3.01 f 0.29 20:4n6 I I .34 + 0.90 20:5n3 0.72 ? 0.06 22:4n6 0.56 + 0.07 22:5n6 0.68 + 0.14 22:Sn3 0.84 ?r 0.08 22:6n3 2.91 -I 0.28
27.30 f 12.30 f 14.43 f 22.20 f 0.00 * 0. I8 & 3.26 ? I I .x0 f 0.69 k 0.40 * 0.33 * 0.90 t 3. IS *
RBC /oral phospholipids l6:O 20.3 I k 0.26 IS.72 * 0.5 I l8:O i&In9 17.03 * 0.48 18:2n6 10.98 f 0.20 IX:3nh 0.00 f 0.00 I8:3n3 0.18 f 0.02 I.71 f 0.02 20:3nh 20:4nh 16.76 f 0.21 20:5n3 0.62 f 0.03 22:4nh 3.65 f 0. I3 22:5n6 1.61 f0.17 22:5n3 2.24 + 0.08 22:6n3 4.01 f 0.08
20.60 + 0.5X 15.85 f I .02 18. IX f 0.36** I I .68 k 0.29** 0.00 * 0.00 0.01 I!zo.O4** I .58 + 0.03** 17.63 + 0.44** 0.59 * 0.03* 3.40 f 0.07** 0.99 * 0. IO** 2.43 + 0.06** 4.36 + 0. I2**
0.37** 0.66 0.15 0.38 0.00 0.02** 0. IO* 0.20 0.02 0.04** 0.06** 0.02*
0.04*
Group 3
Group 4
Group 5
27.68 ? 0.47** 13.37 * 0.24* 14.9s * 0.24 22.9 I * 0.52 0.0I * 0.05 0.14 + 0.07** 2.76 f O.O7* Il.l6f0.12 0.6X f 0.0 I 0.42 f 0.05** 0.42 f O.lh** 0.79 I? 0.03 2.82 f 0.05
27.59 +0.57** 13.6 I + 0.29* 14.77 & 0.28 2 I .S I ? 0.36** o.OO & 0.00 0. I Y * 0.03* 2.99 IL 0.14 I I.2.5 + 0.30 0.66 * 0.03** 0.46 * 0.08** 0.50 f 0.14** 0.83 + 0.04 2.99 f 0. I2
1x.54*2.11** IS.68 i 0.45** IS.74 * 0.25 1-J 31 + 0.64 --.. 0.00 ?zMM) 0. I9 * o.oh* 3.‘) I + 0.32** 13.77 * 0.73** 0.x.3 & o.o’)** OS8 ?r 0. IO 0.63 + 0. I2 I .03 + 0.06** 3.84 * 0.2.5**
2 I .6S 15.44 17.2 I I I .3s 0.00 0.12 I .62 16.28 0.59 3.38 I .48 2.08 3.78
20.64 16.20 17.X9 I I .40 0.00 0. I I I .6S 17.04 0.59 3.50 I .3Y 2.1’) 4.0 I
17.62 13.43 17.54 12.56 (I.00 0.2 I I .Y7 IV.86 0.90 3.73 I .hl 2.x4 5. I6
The results are expressed as mean + SD. * Significantly different from the baseline group al p < 0.05 ** Significantly different from the baseline group at p < 0.01
+ + f * * * ? + * * * * f
0.39** 0.56 0.2x 0.20** 0.00 0.07* 0. IO* 0.39** 0.04 0.17** 0.25 0.08** 0.16**
* + f f f + f * * + + * i
0.7 I 0.80 0.61** 0.34** 0.00 0.07** 0. I2 0.3 I * 0.05 0.09** 0.10** 0.06 0. IO
f + + + f * f & * * ? * +
I .02** I .43** 0.43* 0.66** o.oft 0.05 0.1 o** 0.93** 0.2 I * 0.3 I 0.35 0.09** 0.X**
Effect of Heat Inactivation
and Freezing on Fatty Acid Composition
Table 3 Plasma triglyceride and cholesterol ester composition (initial study) (results expressed as area percent)
Group 1 Plasma triglwerides 16:O 22.88 * 0.38 18:O 3.61 k 0.17
18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3 Plasnta 16:0 18:O
18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3
44.01 * 1.01 17.19 i 1.41 0.34 * 0.19 0.98 k 0.05 0.18kO.11 1.18 f 0.20 0.00 + 0.00 0.00 f 0.00 0.00 f 0.00 0.21 i 0.1 I 0.27 f 0.14 cholesterol
23.20 f 0.18* 3.22 f 0.25** 43.12 f 0.49* 17.25kO.46 0.40 f 0.09
1.OOf 0.06 0.17 f 0.02 1.08 + 0.04 0.00 f 0.00 0.00 + 0.00 0.00 f 0.00 0.14f0.13 0.31 * 0.10
23.48 3.86 44.71 16.90 0.21 0.82 0.15 1.13 0.03 0.00 0.00 0.00 0.14
205
before and after freezing and heat inactivation
Group 3
Group 2
of Plasma and Red Blood Cells
f + * f * + + * + * -t i *
0.3 I** 0.12** 1.11 0.52 0.15 0.26 0.07 0.10 0.07 0.00 0.00 o.oo** 0.17
Group 4
Group 5
23.50 3.67 44.04 16.65 0.33 0.89 0.15 I .06 0.00 0.00 0.00 0.00 0.16
* 0.40** k 0.07 k 0.27 + 0.15 f 0.08 + 0.07** f 0.05 f 0.04 f 0.00 rt 0.00 a? 0.00 + o.oo** f 0.16
14.26 4.98 52.92 19.78 0.18 1.08 0.15 1.54 0.00 0.00 0.00 0.03 0.08
f i. k i f f f + f f + k f
2.93** 0.47** 3.41** 1.37** 0.35 0.06** 0.10 0.18** 0.00 0.00 0.00 0.09** 0.17**
11.23 1.20 20.51 50.99 0.95 0.36 0.64 7.87 0.68 0.00 0.00 0.00 0.72
k 5 k k r f + f f f + + +
6.02 1.82 21.28 52.88 0.79 0.47 0.90 10.59 0.86 0.01 0.00 0.00 I .20
+ k f k i * k * f f + + f
2.97** 0.39** 0.97** 1.55* 0.08** 0.13 0.15** 2.28** 0.29 0.04 0.00 0.00 0.76**
esters
10.97 f 0.48 1.01 kO.11 20.18 k 0.53 5 I .37 + 0.77 1.10~0.12 0.44 + 0.06 0.65 + 0.02 7.61 k 0.20 0.70 k 0.05 0.00 k 0.00 0.00 f 0.00 0.00 + 0.00 0.48 f 0.02
10.32 1.01 19.80 51.59 1.13 0.48 0.73 8.03 0.71 0.00 0.00 0.00 0.52
f 0.81* f 0.09 f 1.10 f 1.14 kO.08 + 0.06 f 0.07** + 0.63 f 0.06 f 0.00 f 0.00 + 0.00 k 0.18
11.48f 0.75 1.41 f 0.77 20.36 + 0.91 50.41 k 2.36 0.90 i 0. IO** 0.36fO.11 0.62 k 0.02** 7.83 + 0.28 0.68 f 0.05 0.02 f 0.07 0.02 + 0.06 0.00 +_0.00 0.83 f 0.42*
0.45 0.38 0.31
1.04 0.14* 0.06* 0.03 0.22* 0.05 0.00 0.00 0.00 0.20**
The results are expressed as mean + SD. * Significantly different from the baseline group at p < 0.05 ** Significantly different from the baseline group at p < 0.01
samples which were incubated at 55 “C, frozen for 1 week and reheated at 55 “C (group 5) showed the greatest significant difference compared to baseline. The percentages of samples per group differing from baseline by less than 5%, between 5.1-9.9%, between lO-19.9% or greater than 20% were calculated from the fatty acid results. While groups 2-4 showed significant differences, few showed changes greater than 10% in any fatty acid when compared to corresponding baseline samples. Group 5 showed differences greater than 10% and often greater than 40%. The results of the second study are shown in Tables 4 and 5. As seen in Table 4, there are some significant changes in fatty acid levels in plasma and RBC phospholipids when compared to baseline. As seen in Table 5, there are also significant changes in plasma triglyceride and cholesterol esters.
DISCUSSION It is now well known that treatment of blood suspected of harbouring HIV and other retroviruses with temperatures of 55-60 “C for 30 min or more will completely destroy the infectivity of the virus present (8-14). Because this procedure is common and in fact advised, and because the effect of this treatment on fatty acid levels has not been determined, we compared the fatty acid levels in RBC phospholipids; and plasma phospholipids,
triglycerides and cholesterol esters to study changes observed after various treatment regimens involving heating and freezing. In the initial study, there were changes in fatty acid levels observed in all fractions studied when compared to baseline samples. Previous work done in our laboratory has shown that plasma and RBC fatty acid values change after storage at 4 “C for as little as 2 h. In that study, after the 2-h period, RBC levels showed significant but small changes from baseline in every fatty acid examined with the exception of palmitic and oleic acids (unpublished results). The small changes (less than 10%) seen in fatty acid levels in all groups in this study may reflect this almost immediate storage effect. The fatty acids of blood samples which were incubated at 55 “C, frozen and then reheated (group 5) showed the largest change from initial values (group 1). A high percentage of these fatty acid values differed from controls by more than 20% and some by as much as 40%. The results from the second experiment showed significant changes in fatty acid patterns after freezing followed by heat inactivation when compared to baseline values. In an unpublished study, we treated plasma and RBCs immediately upon separation with an antioxidant butylated hydroxy-toluene (BHT), to ascertain if the presence of an antioxidant could decrease the degree of change in fatty acid levels after freezing and heat inactivation. Solubility of BHT in plasma and RBC proved to be a problem. Fatty acid levels of plasma and RBCs
206
Prostaglandins
Leukotrienes
and Essential Fatty Acids
Table 4 Total phospholipid composition of RBC and plasma before and after freezing and heat inactivation (second study) (results expressed as area percent) Baseline
Freezing and incubation
Plasma total pl~ospl~olipids
16:O 18:O 18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3
25.40 11.38 14.58 24.46 0.00 0.26 3.06 12.13 0.00 0.49 0.52 0.76 3.32
? 0.61 + 1.46 * 0.73 + 1.86 z+0.00 * 0.03 F 0.35 !c 0.93 f 0.00 f 0.10 f 0.12 f 0.03 f 0.25
Table 5 Plasma triglyceride and cholesterol ester composition before and after freezing and heat inactivation (second study) (result\ expressed as area percent)
26.33 12.41 16.77 23.57 0.00 0.14 2.99 10.12 0.75 0.34 0.30 0.65 2.85
? + * * * * i f ? + * f f
0.61 0.70 0.22** 0.47 0.00 0.02** 0.15 0.35** 0.08** 0.06* 0.19** 0.06** 1.23
Baseline P/usrm~ tr-i,q/yerides 16:O
18:O 18:ln9 18:3n6 IX:3n6 18:3n3 30:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 226n.3 Phsnw
RBC rota1 p~tosp~oiipids l&O 18:0
20.58 + 0.43 12.81 k 0.29
21.42i 1.08* 15.08 k 0.63**
16:O
18:ln9 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 22:4n6 22:5n6 22:5n3 22:6n3
16.76 13.16 0.00 0.21 1.86 16.38 0.00 3.34 1.25 2.10 4.69
18.45 11.40 0.00 0.18 1.48 14.36 0.62 3.43 1.47 1.88 4.09
18:lnY 18:2n6 18:3n6 18:3n3 20:3n6 20:4n6 20:5n3 ?2:4n6 22:5n6 22:5n3 22:6n3
f + f * + f * * f + *
0.19 0.27 0.00 0.01 0.02 0.51 0.00 0.07 0.06 0.06 0.17
f f * * f * f + f f *
0.78** 0.53** 0.00 0.08 0.21** 1.23** 0. IO** 0.23 0.19** 0.18** 0.34**
The results are expressed as mean + SD. * Significantly different from the baseline group at p < 0.05 ** Significantly different from the baseline group at p < 0.01
analysed immediately, and after freezing and heat inactivation, were compared. It was found that the antioxidant was ineffective at preventing changes in fatty acid values. We conclude that RBCs and plasma fatty acid levels are not substantially affected by a 3-h incubation at 55 “C, or by freezing at -20 “C for a period of 1 week or even by these two treatments combined. However we do conclude that a second incubation, after the initial heating and freezing, changes the fatty acid values so that they do not accurately reflect the composition of fatty acids in the blood of patients. We also conclude that heat inactivation following an initial freezing yields unsatisfactory results. From these results, it is clear that blood samples suspected of being contaminated with the HIV virus or other retroviruses can be treated with a 3-h incubation at 55 “C before any freezing and shipment occur, without adverse effects on the fatty acid composition. References 1. Rocklin R. Manku MS, Morse N. Plasma, red blood cell and white cell phospholipid essential fatty acids in atopic subjects with respiratory symptoms. Prog Lipid Res 1986: 25: 203-204 2. Manku MS. Hoi-robin DF, Morse NL, Morse NL, Wright S, Burton JL. Essential fatty acids in the plasma phospholipids of patients with atopic eczema. Br J Dermatol 1984: 110 (6): 643-648
I8:O
cholesteroi
22.18 -t 0.62 3.43 + 0.13 43.21 f 0.36 17.17f0.16 0.30 f 0.04 1.09 f 0.05 0.1’20.09 I .08 f 0.06 0.00 * 0.00 0.00 * 0.00 0.00 * 0.00 0.00 * 0.00 0.52 * 0.02
Freezing and incubation
22.27 3.26 45.34 15.77 0.23 0.84 0.12 0.71 0.14 0.07 0.00 0.15 0.38
f f + * * + * * i: + t * +
0.68 0. IJ* 0.44** 0.86** 0.15 0.0X*” 0.05 0.03** 0.02** 0.02** 0.00 0.01** 0.0 I **
esteiJ
10.12 0.84 20.09 54.30
f f f + 1.06f 0.52 It 0.62 f 6.91 f 0.00 f 0.00 * 0.00 + 0.00 * 0.45 *
0.28 0.08 1.Oh 0.89 0.08 0.05 0.03 0.30 0.00 0.00 0.00 0.00 0.06
11.01f ()..&$“” 1.00 2 I .77 53.13 0.99 0.48 0.53 5.44 0.42 0.00 0.00 0.00 0.28
* * f * * + + * * * + +
0.13** 0.53* 1.40* 0.20 0.07 0.05** 0.4 1x* 0.12** 0.00 0.00 0.00 0. I I **
The results are expressed as mean f SD. * Significantly different from the baseline group at p < 0.05 ** Significantly different from the baseline group at p < 0.01
3. Manku MS. Horrobin DF. Huang YS. Morse N. Fatty acids in plasma and red cell membranes in normal humans. Lipids 1983: 18(12): 906908 4. Hotrobin DF, Ells K, Morse-Fisher N. Manku MS. Fatty acid distribution in plasma phospholipids in normal individuals from different geographical locations. J Nutr Med 1991: 249-258 5. Wright S. Morse N. Manku MS. Essential fatty acids in plasma of patients with leprosy. Int J Lepr 1991; 59(2): 271-277 6. Thuluvath PJ. Triger DR, Manku MS, Morse-Fisher N. Evening primrose oil in the treatment of severe refractory biliary pruritis. Eur J Gastroent Hepatol 1991: 3: X7-90 7. Begin ME, Manku MS. Horrobin DF. Plasma fatty acid levels in patients with acquired immune deficiency syndrome and in controls. Prostaglandins Leukot Essent Fatty Acids 1989; 37: 135-I 37 8. Spire B, Dormont D, Barre-Sinoussi F. Montagnier L. Chermann JC. Inactivation of lymphadenapathyassociated virus by heat, gamma rays and ultraviolet light. Lancet 1985: i: 188-189 Ginosa W. Inactivation of viruses by ionizing radiation\ and by heat. In: Maramorosch K. Koprowski H (eds). Methods in virology IV. New York: Academic Press. 1968: 139-209 McDougal JS. Martin LS. Con SP. Mozen M. Heldebrant CM, Evatt BL. Thermal inactivation of acquired immunodeficiency syndrome virus, human T lymphotropic virus-III/lymphadenopathy-associated virus. with special reference to antihemophilic factor. J Clin Invest 1985; 76: X75-877 Dolana G. Hollinger FB. Thomas W. Gyorkey F. Continued observations on the effect of a heating procedure on the inactivation of non-A, non-B hepatitis and hepatitis B viruses in clotting factor concentrate. Thromb Haemost 1983; 50: 115 Martin LS, McDougal JS. Loskoski SL. Disinfection and
Effect of Heat Inactivation inactivation of the human T lymphotropic virus type III/ lymphadenopathy-associated virus. J Infect Dis 1985: 152 (2): 4wo3 13. Klein M, Deforest A. Principles of viral inactivation. In: Block SS (ed) Disinfection, sterilization, and preservation 3rd ed. Philadelphia: Lea and Febiger 1983; 422434 14. Einarsson M, Perenius L, McDougal JS. Cort S. Heat inactivation of human immunodeficiency virus in
and Freezing on Fatty Acid Composition
of Plasma and Red Blood Cells
solutions of antithrombin III. Transfusion 1989; 229(2): 148-152 15. Folch J. Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957: 226:497 16. Manku MS, Horrobin DF, Huang YS, Morse N. Fatty acids in plasma and red cell membranes in normal humans. Lipids 1983; 18(12): 906908
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