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Stability of the long chain non-esterified fatty acid pattern in plasma and blood during different storage conditions
49
Clinica Chimica Acta,
@ Elsevier/North-Holland
84
(1978) 49-54 Biomedical
Press
CCA 9074
STABILITY OF THE LONG CHAIN NON-ESTERIFIED FATTY ACID PATTERN IN PLASMA AND BLOOD DURING DIFFERENT STORAGE CONDITIONS
VERA ROGIERS Lubomtorium voor Fysiologische Scheikunde, Vrije Universiteit Brussel, Brussel (Belgium) (Received
Faculteit
Geneeskunde
en Farmacie,
July 22nd, 1977)
summary A study has been carried out to determine the conditions under which plasma and blood can be stored without significant changes in the non-esterified fatty acid (NEFA) pattern and in the total NEFA concentration. From the results obtained it appears that: (1) blood can be stored for 2 h at room temperature and for 48 h at 4°C. (2) plasma can be stored for 6 h at room temperature, 48 h at 4°C and for at least 10 days at -20°C under nitrogen without significant changes in the NEFA pattern and in the total NEFA concentration. The possible influence of heparin, used as anticoagulant, has been investigated. The results obtained are compared with those of other investigators.
Introduction Several studies have been done to determine the stability of the total nonesterified fatty acid (NEFA) concentration in plasma and blood during different storage conditions [l-9]. Many contradictory results were published and usually no information is given about the stability of the NEFA pattern. As the general belief is that the total NEFA concentration of plasma and blood changes during storage at room temperature (Tn), the blood is analysed at once after collection. From a practical point of view, in research as well as in clinical studies of the NEFA pattern, it appears desirable to store a number of plasma samples in order to analyse them simultaneously at a more convenient time. This study has been undertaken to determine the conditions under which
50
blood and plasma can be stored without tern and in the total NEFA concentration.
significant
changes in the NEFA pat-
Materials and methods Reagent as described previously [lo]. All tubes and vessels, used for storage of plasma and blood samples, are made of glass. Methods The plasma NEFA pattern and the total NEFA concentration have been determined by the improved gas-liquid chromatographic method which we described earlier [lo]. Storage of blood at TR (23-25”C), 4°C and 37°C. Blood was collected into an ice-cooled heparinised (100 I.U./ml blood) vessel and distributed in different screw-top tubes after mixing gently. A control determination was carried out immediately and the results obtained were taken as the 100% values. The remaining tubes were stored for 24 h at T,, for 96 h at 4°C and for 1 h at 37”C, before the blood was centrifuged at 4°C and the NEFA’s quantitated. Storage of plasma at TR (23-25”C), 4°C and -20°C. Blood was collected into an ice-cooled heparinised vessel, mixed gently and centrifuged immediately at 4°C. The plasma was promptly transferred to different screw-top tubes and a control determination was carried out immediately (100% value). The remaining tubes were stored for 24 h at T,, for 96 h at 4°C and for 30 days at -2O”C, before the plasma NEFA pattern was determined by the GLC method [lo]. Through the plasma samples, stored at -2O”C, pure nitrogen was bubbled for 1 min, carefully to avoid frothing. Each sample was frozen only once. Influence (in vitro) of heparin. Blood was taken into ice-cooled tubes containing heparin in different concentrations (Table II). After centrifugation at 4°C a control determination (heparin content of 100 I.U./ml blood) was carried out at once. The remaining tubes were stored for 2 h at TR before they were analysed. All experiments were carried out five times in triplicate on blood of various apparently healthy adults (between 25 and 35 years old), who were fasting from 10 p.m. until blood collection at 9 a.m. (none of them was hyperlipemic). Results Storage of blood at TR, 4°C and 37°C The stability of the total NEFA concentration of blood kept at TR and 4”C, is shown in Fig. 1. Blood can be stored for 2 h at TR and for 48 h at 4”C, without significant changes in the total NEFA concentration and in the NEFA pattern. After 3 and 4 h at TR, the total NEFA concentration still remains constant, but the NEFA pattern changes. After a storage of 24 h at TR, the changes of the NEFA pattern are very significant: for the unsaturated fatty acids as palmitoleic (C16: ,), linoleic (ClaEa) and linolenic acid (C18: 3), the % of the control values is two times higher than for the saturated stearic acid (C,,,,).
51
__ -.]-____ f It T-i---’ 1_ _ _ _ _ _ _ _ _ _ _ _ _ _ ’I
T
T
1
T
b
9OJ , : , , , , 0123456 Ttme ot storage
, , , ,
8 ( hours
10
>
)
Fig. 1. Effect of storage on the total NEFA concentration of blood; ‘Methods. Curve a, blood kept at TR: curve b. blood kept at 4’C.
working
conditions
as described
in
Increases in the NEFA content of blood immediately after blood collection, as mentioned by several investigators [ 1,3,5,7], have never been found in any of the analysed samples. At 4°C the NEFA content of the blood samples changes only after 48 h: here again the Clszl and C1sz3 levels increase more than those of the remaining NEFA’s. After 1 h storage of the blood at 37”C, considerable changes have been noticed in the total NEFA concentration (increase of 20%) as well as in the NEFA pattern. Therefore it is advisable to cool the blood immediately after collection. Storage of plasma at TR, 4°C and -20°C The results obtained for the total NEFA concentration of plasma, stored at TR and 4”C, are shown in Fig. 2. Plasma, prepared and analysed as described in Methods, can be stored for 6 h at TR, without significant changes in the total NEFA concentration and in the
a
; f ,;;j
1f Time
//’ $y?L~L_________~ of storage
8
(hours
IO
1
Fig. 2. Effect of storage on the total NEFA concentration of plasma; Methods. Curve a. plasma kept as TR: curve b. plasma kept at 4%.
working
conditions
as described
in
52 TABLE I EFFECT OF STORAGE UNDER NITROGEN NEFA CONCENTRATION OF PLASMA
AT -2O’C
ON THE NEFA PATTERN
AND THE TOTAL
Cl4 : 0, myristic acid; Cl6 : 0, pslmitic acid; Cl6 : 1, pabhitoleic acid; Cl8 : 0. stearic acid; Cl8 okic acid: Cl8 : z9 linOkiC acid: Cl8 : CJ* linolenic acid. Figures represent mean ?: S.D. Fatty acid
% of control at -2O’C
: 1,
under N2 (control = 100%)
Time of storage in days 2 Cl4 : 0 c16 : 0 c16 : 1 c18 : 0 c18 : 1 Cl8 : 2 Cl8 : 3 Total NEFA
4
94.5 104.2 99.2 111.7 96.3 91.5 96.7 98.8
12
90.7 97.9 92.2 96.6 101.7 104.5 114.9 100.0
+ 9.9 + 4.3 f 9.8 r 9.9 + 8.6 f 9.7 f 7.1 k 14.0
+ 8.6 f 5.0 fr 10.3 t 8.1 f 3.8 + 2.0 f 7.5 k 4.2
86.9 97.2 103.3 96.2 103.0 99.6 98.6 100.3
?- 10.8 f 6.1 f 10.1 r 6.1 + 9.6 + 1.0 f 6.5 f 7.1
NEFA pattern. After a longer storage period, the individual NEFA concentrations increase disproportionately: both the total NEFA concentration and the NEFA pattern change. After keeping the plasma samples for 24 h at TR, the percentage of the control determination8 for C16:, , C,8:2 and C18:3 is two times higher than for C 1a:o. The plasma samples can be stored at 4°C for at least 48 h, without special TABLE II INFLUENCE THE TOTAL
OF THE HEPARIN CONCENTRATION NEFA CONCENTRATION OF PLASMA
(IN VITRO) ON THE NEFA PATTERN AFTER A 2 h STORAGE AT TR
AND
Figures represent mean (S.D.). % of control (control = 100%)
Fatty acid
Heparin concentration in I.U./ml blood 0.1 Cl4 c16 c16
0.5
1
3
5
10
20
30
99.5 (12.9) 96.4
105.9 (11.2) 106.4
100.5 (15.9) 104.3
(8.4)
(4.4) 101.6 (12.3) 89.8 (11.3) 100.5
(4.5) 114.9 (11.4) 109.5
99.6 (16.8) 100.9 (10.7) 112.4 (12.5) 104.6
(5.0) 101.6
:0
105.2
107.2
: 0
(7.5) 101.1
(4.8) 98.7
:
(0.1) 94.6
(2.8) 99.7
109.8 (10.8) 112.7 (18.4) 109.5
(4.6) 90.5 (12.2) 103.2
(7.2) 99.7 (11.7) 110.4
(0.5) 105.2
(5.3) 99.8
(2.3) 103.4
(8.7) 110.3
1
c18
:
1
(4.5) 88.2 (14.6) 99.8
c18
:
2
(1.4) 99.3
(5.3) 102.6
c18
:3
(1.6) 111.7 (12.3) 98.4
(0.9) 101.1
(4.5) 99.3 (10.3) 103.6
(7.2) 98.3
(9.8) 103.1
(7.5) 97.7
(8.4) 105.4
(1.6)
(4.0)
(7.2)
(1.8)
(3.5)
C18:
0
Total NEFA
(5.7) 99.3 (10.4) 99.3 (8.2) 106.3 (15.6) 101.5 (10.3)
92.9 108.1 (5.8) 107.5
50 89.9 (11.8) 100.9 (13.0) 110.9 (11.6) 97.3
(9.9) 113.0 (12.9) 101.8
(4.9) 103.5
(4.3) 104.9
(3.1) 105.9
(7.4) 105.3 (5.2) 104.5 (14.5) 106.1
(2.5) 107.1 (11.7) 91.8 (16.9) 107.8
(4.3) 96.4 (13.1) 97.3 (11.9) 101.3 (18.9) 99.2
(8.1)
(6.3)
(6.8)
63
precautions: after 96 h the NEFA concentrations rise, especially those of C&s:2 and %: 3. The NEFA pattern and the total NEFA concentration of plasma, stored under nitrogen at -2O”C, do not change within 10 days (Table I). The results of the storage for a,longer period of time than 10 days are not uniform. Very contradictory results have been found for plasmas of different persons. The NEFA content of some plasma samples remained constant, even after 30 days, but further study is necessary for general conclusions to be drawn. Oxidation of the double bonds, especially in C& and C18:3 may occur, when plasma is stored at -20°C without the special precaution of a nitrogen atmosphere. Influence (in vitro) of heparin
The effect (in vitro) of the heparin concentration on the NEFA content of plasma has been investigated and no significant changes have been found when the plasmas have been stored at Ta for 2 h (Table II). Discussion From the results obtained it appears that: (I) Blood may be stored for 2 h at Z’a and for 48 h at 4°C without significant change in the NEFA pattern or in the total NEFA concentration, (2) Plasma can be stored for 6 h at Ta and for 48 h at 4”C, when it is centrifuged and separated immediately after blood collection. The results of Figs. 1 and 2 have been statistically analysed with a x2 test (degrees of freedom = 5). As for every curve &culated < x&, the null hypotheses are not rejectable. Our conclusions about the storage of plasma and blood at Z’n and 4°C are partly in contradiction with those of other investigators, especially the results about the storage at TR [1,3,5,‘7]. In a titrimetric study, Forbes and Camlin mention an increased serum total NEFA concentration of 50% after storage at TR for 1 h [ 11. Howorth et al. [ 31 and Braun [5] observe a continuous rise of the total NEFA concentration, respectively in plasma and in serum during storage at T,, immediately after blood collection, and Sampson and Hensley [‘?I find significant rises in the total NEFA concentration of plasma (22%) and blood (68%) after storage at TR for 1 h. Our findings about the stability of the plasma total NEFA concentration at 4°C agree with those of Sampson and Hensley [7] and Miihlfellner [6], but differ from those of Braun [ 51 and Trichopoulon et al. [S]. Munkner [2] has reported that the plasma NEFA content remained unchanged for only 6 h at 4°C. As each of our determinations has been carried out 5 times in triplicate, it seems unlikely that the discrepancies between our own results and those of other investigators are due to technicaI errors. We have considered a possible in vitro influence of heparin, used as anticoagulant, on the plasma NEFA concentration. As previous studies mention an important rise in the total NEFA concentration already after 2 h storage of
54
plasma at TR, our determination of any heparin influence have been carried out after keeping the plasma samples for 2 h at T,. According to Patelski et al. [ll] heparin would influence (in vitro) the lipolytic enzyme activity of glycerol ester hydrolase in blood serum. The lipase would be activated at low and inhibited at high concentrations of heparin. From our results (Table II) it appears that the heparin concentration does not influence (in vitro) the plasma NEFA pattern nor the total NEFA concentration. Even at very low heparin concentration (0.1 I.U./ml blood), there was no significant rise in the NEFA content of the plasma. Different opinions have been expressed concerning the storage of plasma at -20°C [ 1,3-81. From the results shown in Table I, it appears that the NEFA pattern and the total NEFA concentration of plasma, stored at -20°C under nitrogen, remain constant for 10 days. These findings agree with those of Regouw et al. [4], Braun [ 51, Miihlfeller [6] and Sampson and Hensley [7], but are in contradiction with those of Forbes and Camlin [l] ; Howorth et al. [ 31 and Trichopoulon et al. [ 81. The instability of the NEFA’s in plasma and blood is commonly explained as an enzymatic (lipase and lecithinase) or a physiocochemical action [ 1,3,5,7,8]. From our results it appears that the lower the temperature, the longer plasma and blood can be stored without significant changes in the NEFA pattern and in the total NEFA concentration. As plasma can be stored at TR for 4 h longer than blood without change, enzymatic action may not be excluded as a possible cause for the instability of the NEFA’s during storage of plasma and blood. Acknowledgements This work We wish to and Dr. J.M. Hospital for Mr. J. Dewit
is supported by the Medical Foundation “Koningin Elisabeth”. thank Dr. A. Gallez from the pediatric section (Professor H. Vis) De Steckx from the blood transfusion section of the Saint-Pierre the blood collections, Mr. R. De Beule for his statistical advice and for his technical assistance.
References 1 2 3 4 5 6 7 8 9 10 11
Forbes, A.L. and Camlin, J.A. (1959) Proc. Sot. EXP. Biol. Med., N.Y. 102. 709 Munkner, C. (1959) Scandinav. J. Clin. Lab. Invest. 11, 388 Howorth, P,J.N., Gibbard, S. and Marks, V. (1966) Clin. Chim. Acta 14,69 Regouw. B.J.M., Cornelissen. P.J.H.C., Helder, R.A.P.. Spijkers. J.B.F. and Weeber, Y.M.M. Clin. Chim. Acta 31,187 Braun, J.S. (1971) 2. Klin. Chem.Klin. Biochem. 9,387 Miihlfellner, 0. (1972) Z. Klin. Chem. Klin. Biochem. 10, 37 Sampson, D. and Hensley. W.J. (1975) Clin. Chim. Acta 61,l Trichopoulon, A., Kalaidzidou, C. and Kalandidi. A. (1976) Clin. Chim. Acta 69. 355 Hirsch, E.Z., Slivka, S. and Gibbons, A.P. (1976) Clin. Chem. 22,445 Rogiers, V. (1977) Clin. Chim. Acta 78,227 Patelski. J., Pniewska, B., Szulc. S. and Waligora. Z. (1968) Diagn. Lab. 4,131
(1971)
Clinica Chimica Acta,
@ Elsevier/North-Holland
84
(1978) 49-54 Biomedical
Press
CCA 9074
STABILITY OF THE LONG CHAIN NON-ESTERIFIED FATTY ACID PATTERN IN PLASMA AND BLOOD DURING DIFFERENT STORAGE CONDITIONS
VERA ROGIERS Lubomtorium voor Fysiologische Scheikunde, Vrije Universiteit Brussel, Brussel (Belgium) (Received
Faculteit
Geneeskunde
en Farmacie,
July 22nd, 1977)
summary A study has been carried out to determine the conditions under which plasma and blood can be stored without significant changes in the non-esterified fatty acid (NEFA) pattern and in the total NEFA concentration. From the results obtained it appears that: (1) blood can be stored for 2 h at room temperature and for 48 h at 4°C. (2) plasma can be stored for 6 h at room temperature, 48 h at 4°C and for at least 10 days at -20°C under nitrogen without significant changes in the NEFA pattern and in the total NEFA concentration. The possible influence of heparin, used as anticoagulant, has been investigated. The results obtained are compared with those of other investigators.
Introduction Several studies have been done to determine the stability of the total nonesterified fatty acid (NEFA) concentration in plasma and blood during different storage conditions [l-9]. Many contradictory results were published and usually no information is given about the stability of the NEFA pattern. As the general belief is that the total NEFA concentration of plasma and blood changes during storage at room temperature (Tn), the blood is analysed at once after collection. From a practical point of view, in research as well as in clinical studies of the NEFA pattern, it appears desirable to store a number of plasma samples in order to analyse them simultaneously at a more convenient time. This study has been undertaken to determine the conditions under which
50
blood and plasma can be stored without tern and in the total NEFA concentration.
significant
changes in the NEFA pat-
Materials and methods Reagent as described previously [lo]. All tubes and vessels, used for storage of plasma and blood samples, are made of glass. Methods The plasma NEFA pattern and the total NEFA concentration have been determined by the improved gas-liquid chromatographic method which we described earlier [lo]. Storage of blood at TR (23-25”C), 4°C and 37°C. Blood was collected into an ice-cooled heparinised (100 I.U./ml blood) vessel and distributed in different screw-top tubes after mixing gently. A control determination was carried out immediately and the results obtained were taken as the 100% values. The remaining tubes were stored for 24 h at T,, for 96 h at 4°C and for 1 h at 37”C, before the blood was centrifuged at 4°C and the NEFA’s quantitated. Storage of plasma at TR (23-25”C), 4°C and -20°C. Blood was collected into an ice-cooled heparinised vessel, mixed gently and centrifuged immediately at 4°C. The plasma was promptly transferred to different screw-top tubes and a control determination was carried out immediately (100% value). The remaining tubes were stored for 24 h at T,, for 96 h at 4°C and for 30 days at -2O”C, before the plasma NEFA pattern was determined by the GLC method [lo]. Through the plasma samples, stored at -2O”C, pure nitrogen was bubbled for 1 min, carefully to avoid frothing. Each sample was frozen only once. Influence (in vitro) of heparin. Blood was taken into ice-cooled tubes containing heparin in different concentrations (Table II). After centrifugation at 4°C a control determination (heparin content of 100 I.U./ml blood) was carried out at once. The remaining tubes were stored for 2 h at TR before they were analysed. All experiments were carried out five times in triplicate on blood of various apparently healthy adults (between 25 and 35 years old), who were fasting from 10 p.m. until blood collection at 9 a.m. (none of them was hyperlipemic). Results Storage of blood at TR, 4°C and 37°C The stability of the total NEFA concentration of blood kept at TR and 4”C, is shown in Fig. 1. Blood can be stored for 2 h at TR and for 48 h at 4”C, without significant changes in the total NEFA concentration and in the NEFA pattern. After 3 and 4 h at TR, the total NEFA concentration still remains constant, but the NEFA pattern changes. After a storage of 24 h at TR, the changes of the NEFA pattern are very significant: for the unsaturated fatty acids as palmitoleic (C16: ,), linoleic (ClaEa) and linolenic acid (C18: 3), the % of the control values is two times higher than for the saturated stearic acid (C,,,,).
51
__ -.]-____ f It T-i---’ 1_ _ _ _ _ _ _ _ _ _ _ _ _ _ ’I
T
T
1
T
b
9OJ , : , , , , 0123456 Ttme ot storage
, , , ,
8 ( hours
10
>
)
Fig. 1. Effect of storage on the total NEFA concentration of blood; ‘Methods. Curve a, blood kept at TR: curve b. blood kept at 4’C.
working
conditions
as described
in
Increases in the NEFA content of blood immediately after blood collection, as mentioned by several investigators [ 1,3,5,7], have never been found in any of the analysed samples. At 4°C the NEFA content of the blood samples changes only after 48 h: here again the Clszl and C1sz3 levels increase more than those of the remaining NEFA’s. After 1 h storage of the blood at 37”C, considerable changes have been noticed in the total NEFA concentration (increase of 20%) as well as in the NEFA pattern. Therefore it is advisable to cool the blood immediately after collection. Storage of plasma at TR, 4°C and -20°C The results obtained for the total NEFA concentration of plasma, stored at TR and 4”C, are shown in Fig. 2. Plasma, prepared and analysed as described in Methods, can be stored for 6 h at TR, without significant changes in the total NEFA concentration and in the
a
; f ,;;j
1f Time
//’ $y?L~L_________~ of storage
8
(hours
IO
1
Fig. 2. Effect of storage on the total NEFA concentration of plasma; Methods. Curve a. plasma kept as TR: curve b. plasma kept at 4%.
working
conditions
as described
in
52 TABLE I EFFECT OF STORAGE UNDER NITROGEN NEFA CONCENTRATION OF PLASMA
AT -2O’C
ON THE NEFA PATTERN
AND THE TOTAL
Cl4 : 0, myristic acid; Cl6 : 0, pslmitic acid; Cl6 : 1, pabhitoleic acid; Cl8 : 0. stearic acid; Cl8 okic acid: Cl8 : z9 linOkiC acid: Cl8 : CJ* linolenic acid. Figures represent mean ?: S.D. Fatty acid
% of control at -2O’C
: 1,
under N2 (control = 100%)
Time of storage in days 2 Cl4 : 0 c16 : 0 c16 : 1 c18 : 0 c18 : 1 Cl8 : 2 Cl8 : 3 Total NEFA
4
94.5 104.2 99.2 111.7 96.3 91.5 96.7 98.8
12
90.7 97.9 92.2 96.6 101.7 104.5 114.9 100.0
+ 9.9 + 4.3 f 9.8 r 9.9 + 8.6 f 9.7 f 7.1 k 14.0
+ 8.6 f 5.0 fr 10.3 t 8.1 f 3.8 + 2.0 f 7.5 k 4.2
86.9 97.2 103.3 96.2 103.0 99.6 98.6 100.3
?- 10.8 f 6.1 f 10.1 r 6.1 + 9.6 + 1.0 f 6.5 f 7.1
NEFA pattern. After a longer storage period, the individual NEFA concentrations increase disproportionately: both the total NEFA concentration and the NEFA pattern change. After keeping the plasma samples for 24 h at TR, the percentage of the control determination8 for C16:, , C,8:2 and C18:3 is two times higher than for C 1a:o. The plasma samples can be stored at 4°C for at least 48 h, without special TABLE II INFLUENCE THE TOTAL
OF THE HEPARIN CONCENTRATION NEFA CONCENTRATION OF PLASMA
(IN VITRO) ON THE NEFA PATTERN AFTER A 2 h STORAGE AT TR
AND
Figures represent mean (S.D.). % of control (control = 100%)
Fatty acid
Heparin concentration in I.U./ml blood 0.1 Cl4 c16 c16
0.5
1
3
5
10
20
30
99.5 (12.9) 96.4
105.9 (11.2) 106.4
100.5 (15.9) 104.3
(8.4)
(4.4) 101.6 (12.3) 89.8 (11.3) 100.5
(4.5) 114.9 (11.4) 109.5
99.6 (16.8) 100.9 (10.7) 112.4 (12.5) 104.6
(5.0) 101.6
:0
105.2
107.2
: 0
(7.5) 101.1
(4.8) 98.7
:
(0.1) 94.6
(2.8) 99.7
109.8 (10.8) 112.7 (18.4) 109.5
(4.6) 90.5 (12.2) 103.2
(7.2) 99.7 (11.7) 110.4
(0.5) 105.2
(5.3) 99.8
(2.3) 103.4
(8.7) 110.3
1
c18
:
1
(4.5) 88.2 (14.6) 99.8
c18
:
2
(1.4) 99.3
(5.3) 102.6
c18
:3
(1.6) 111.7 (12.3) 98.4
(0.9) 101.1
(4.5) 99.3 (10.3) 103.6
(7.2) 98.3
(9.8) 103.1
(7.5) 97.7
(8.4) 105.4
(1.6)
(4.0)
(7.2)
(1.8)
(3.5)
C18:
0
Total NEFA
(5.7) 99.3 (10.4) 99.3 (8.2) 106.3 (15.6) 101.5 (10.3)
92.9 108.1 (5.8) 107.5
50 89.9 (11.8) 100.9 (13.0) 110.9 (11.6) 97.3
(9.9) 113.0 (12.9) 101.8
(4.9) 103.5
(4.3) 104.9
(3.1) 105.9
(7.4) 105.3 (5.2) 104.5 (14.5) 106.1
(2.5) 107.1 (11.7) 91.8 (16.9) 107.8
(4.3) 96.4 (13.1) 97.3 (11.9) 101.3 (18.9) 99.2
(8.1)
(6.3)
(6.8)
63
precautions: after 96 h the NEFA concentrations rise, especially those of C&s:2 and %: 3. The NEFA pattern and the total NEFA concentration of plasma, stored under nitrogen at -2O”C, do not change within 10 days (Table I). The results of the storage for a,longer period of time than 10 days are not uniform. Very contradictory results have been found for plasmas of different persons. The NEFA content of some plasma samples remained constant, even after 30 days, but further study is necessary for general conclusions to be drawn. Oxidation of the double bonds, especially in C& and C18:3 may occur, when plasma is stored at -20°C without the special precaution of a nitrogen atmosphere. Influence (in vitro) of heparin
The effect (in vitro) of the heparin concentration on the NEFA content of plasma has been investigated and no significant changes have been found when the plasmas have been stored at Ta for 2 h (Table II). Discussion From the results obtained it appears that: (I) Blood may be stored for 2 h at Z’a and for 48 h at 4°C without significant change in the NEFA pattern or in the total NEFA concentration, (2) Plasma can be stored for 6 h at Ta and for 48 h at 4”C, when it is centrifuged and separated immediately after blood collection. The results of Figs. 1 and 2 have been statistically analysed with a x2 test (degrees of freedom = 5). As for every curve &culated < x&, the null hypotheses are not rejectable. Our conclusions about the storage of plasma and blood at Z’n and 4°C are partly in contradiction with those of other investigators, especially the results about the storage at TR [1,3,5,‘7]. In a titrimetric study, Forbes and Camlin mention an increased serum total NEFA concentration of 50% after storage at TR for 1 h [ 11. Howorth et al. [ 31 and Braun [5] observe a continuous rise of the total NEFA concentration, respectively in plasma and in serum during storage at T,, immediately after blood collection, and Sampson and Hensley [‘?I find significant rises in the total NEFA concentration of plasma (22%) and blood (68%) after storage at TR for 1 h. Our findings about the stability of the plasma total NEFA concentration at 4°C agree with those of Sampson and Hensley [7] and Miihlfellner [6], but differ from those of Braun [ 51 and Trichopoulon et al. [S]. Munkner [2] has reported that the plasma NEFA content remained unchanged for only 6 h at 4°C. As each of our determinations has been carried out 5 times in triplicate, it seems unlikely that the discrepancies between our own results and those of other investigators are due to technicaI errors. We have considered a possible in vitro influence of heparin, used as anticoagulant, on the plasma NEFA concentration. As previous studies mention an important rise in the total NEFA concentration already after 2 h storage of
54
plasma at TR, our determination of any heparin influence have been carried out after keeping the plasma samples for 2 h at T,. According to Patelski et al. [ll] heparin would influence (in vitro) the lipolytic enzyme activity of glycerol ester hydrolase in blood serum. The lipase would be activated at low and inhibited at high concentrations of heparin. From our results (Table II) it appears that the heparin concentration does not influence (in vitro) the plasma NEFA pattern nor the total NEFA concentration. Even at very low heparin concentration (0.1 I.U./ml blood), there was no significant rise in the NEFA content of the plasma. Different opinions have been expressed concerning the storage of plasma at -20°C [ 1,3-81. From the results shown in Table I, it appears that the NEFA pattern and the total NEFA concentration of plasma, stored at -20°C under nitrogen, remain constant for 10 days. These findings agree with those of Regouw et al. [4], Braun [ 51, Miihlfeller [6] and Sampson and Hensley [7], but are in contradiction with those of Forbes and Camlin [l] ; Howorth et al. [ 31 and Trichopoulon et al. [ 81. The instability of the NEFA’s in plasma and blood is commonly explained as an enzymatic (lipase and lecithinase) or a physiocochemical action [ 1,3,5,7,8]. From our results it appears that the lower the temperature, the longer plasma and blood can be stored without significant changes in the NEFA pattern and in the total NEFA concentration. As plasma can be stored at TR for 4 h longer than blood without change, enzymatic action may not be excluded as a possible cause for the instability of the NEFA’s during storage of plasma and blood. Acknowledgements This work We wish to and Dr. J.M. Hospital for Mr. J. Dewit
is supported by the Medical Foundation “Koningin Elisabeth”. thank Dr. A. Gallez from the pediatric section (Professor H. Vis) De Steckx from the blood transfusion section of the Saint-Pierre the blood collections, Mr. R. De Beule for his statistical advice and for his technical assistance.
References 1 2 3 4 5 6 7 8 9 10 11
Forbes, A.L. and Camlin, J.A. (1959) Proc. Sot. EXP. Biol. Med., N.Y. 102. 709 Munkner, C. (1959) Scandinav. J. Clin. Lab. Invest. 11, 388 Howorth, P,J.N., Gibbard, S. and Marks, V. (1966) Clin. Chim. Acta 14,69 Regouw. B.J.M., Cornelissen. P.J.H.C., Helder, R.A.P.. Spijkers. J.B.F. and Weeber, Y.M.M. Clin. Chim. Acta 31,187 Braun, J.S. (1971) 2. Klin. Chem.Klin. Biochem. 9,387 Miihlfellner, 0. (1972) Z. Klin. Chem. Klin. Biochem. 10, 37 Sampson, D. and Hensley. W.J. (1975) Clin. Chim. Acta 61,l Trichopoulon, A., Kalaidzidou, C. and Kalandidi. A. (1976) Clin. Chim. Acta 69. 355 Hirsch, E.Z., Slivka, S. and Gibbons, A.P. (1976) Clin. Chem. 22,445 Rogiers, V. (1977) Clin. Chim. Acta 78,227 Patelski. J., Pniewska, B., Szulc. S. and Waligora. Z. (1968) Diagn. Lab. 4,131
(1971)