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Effect of heparin and storage on human plasma free fatty acid concentration
C[inico Chimica Acfu. 169 (1987) 315-318 Elsevier
315
CCA 03994
Short communication
Effect of heparin and storage on human plasma free fatty acid concentration Michael Gleeson Department of Environmental and Occupational Medicine, University Medical School, Aberdeen (UK)
(Received 28 January 1987; revision received 19 June 1987: accepted after revision 3 August 1987) Key words: Plasma; Free fatty acid: Heparin
Introduction In plasma and serum samples obtained from man and other mammals, spontaneous hydrolysis of esterified fatty acids into free fatty acids (FFA) has been reported [l-6]. Several investigators have described the effects of different storage conditions on plasma FFA concentration [1,3-61. This in vitro lipolysis may be of enzymatic or of physico-chemical origin [4] but it is reasonable to postulate that such lipolysis is in part due to the action of lipoprotein lipase [1,2]. Since heparin is known to activate lipoprotein lipase [2] the present study was undertaken to investigate the effect of using heparin as an anticoagulant on the concentration of FFA in human plasma stored at different temperatures. This study was prompted by the recent appearance of two papers in the literature [7,8] in which the investigators used heparin as anticoagulant for human blood and whose reported values of plasma FFA concentrations during exercise were very much higher than those described elsewhere; indeed, their values appear to be close to or above the upper concentration limit which is probably set by the plasma albumin-binding capacity [9,10]. Methods Two series of experiments were conducted. In the first, blood was obtained by venepuncture from 6 healthy adult men at rest after an overnight fast. The blood samples were collected in a dry syringe and portions were added to tubes containing Lithium Heparinate (20 U/ml blood), Potassium EDTA (1.5 mg/ml blood) or no anticoagulant. The blood in the tubes without anticoagulant was allowed to clot for 30 min and the sera separated by centrifugation (2 500 x g for 10 min at 20’ C).
Correspondence to: Dr. Michael Gleeson, Department of Environmental University Medical School, Foresterhill, Aberdeen AB9 2ZD, UK.
and Occupational Medicine,
0009-8981/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
316
From the other tubes, plasma was obtained without delay by centrifugation (2 500 x g for 10 min at 20 o C). FFA [ll] and triglyceride [12] concentrations were determined in all samples just after collection. Triplicate portions of the same plasma or serum were stored at 37, 20, 4 and -20°C and were subsequently analysed for FFA [ll] up to 36 days after collection. In the second series of experiments, serum was obtained from 4 healthy adult males as described above. The serum samples were pooled and immediately analysed for FFA [ll]. The pooled sera were divided into 1.0 ml portions, to which 50 ~1 of saline (control) or solutions of different concentrations of sodium heparinate in saline (155 mmol/l) were added to give final heparin concentrations of 0, 0.5, 2, 10, 20, 50, 100, 200 and 500 U/ml serum. The sera were incubated for 24 h at 37” and the FFA determined [ll]. Results and discussion
The results of the experiments conducted are shown in Figs. 1 and 2. FFA concentration in the samples increased with storage time and increasing the storage temperature augmented these rises (Fig. 1). At all temperatures the increase in plasma FFA was greater when heparin was used as anticoagulant compared with EDTA or no anticoagulant. After 36 days storage at - 20 o C, FFA concentrations were unchanged (compared with initial values) in serum and in plasma anticoagu-
1.0
-
0.8
-
0.6
-
4 IA. IJ.
0.4
-
E ii =
0.2
-
= 3 5E
o0
2
4
6
8
10
12
”
36
Days
Fig. 1. Influence of storage conditions on free fatty acid (FFA) concentration of human plasma using KsEDTA (open symbols) or lithium-heparin (closed symbols) as anticoagulant. Plasma was stored at + 37 o C (v, v), + 20 ’ C (0, l), + 4O C (0, W) and - 20 o C (A, A). Each point represents mean values of triplicate plasma samples from six resting postabsorptive men. FFA values for serum (not shown here) were not significantly different from those obtained with plasma containing KsEDTA. The initial FFA concentration (mean* SD) of the samples was 0.21 kO.07 (range 0.12-0.33) mmol/l and the initial triglyceride
concentration
was 0.83 f 0.25 (range 0.60-1.25)
mmol/l.
317
I
I
0
100
I 200
I
300
Heparin concentration
I
400 (U/ml
l 500 serum
)
Fig. 2. Influence of heparin concentration on the change in human serum free fatty acid (FFA) concentration after 24 h incubation at + 37’ C. Each point represents the mean of triplicate measurements at each heparin concentration. The initial FFA concentration of the pooled sera was 0.26 mmol/l.
lated with EDTA. However, in plasma containing heparin, FFA were 60% higher than initial values (Fig. 1) Figure 2 demonstrates that a wide range of heparin concentrations (OS-500 U/ml) increases the lipolytic activity of serum. Different opinions have been expressed concerning low-temperature storage of plasma or serum samples for subsequent analysis of FFA. Gordon et al [13] used oxalate as anticoagulant and showed that FFA levels do not change when plasma is stored at -20°C. In an earlier study, Forbes and Camlin [l] reported a steadily increasing FFA concentration in serum stored at -20” C. However, in the latter study FFA were analysed by the method of Dole 1141and most of the rise found by Forbes and Camlin [l] may be attributed to titration of non-FFA organic acids originating during storage [3]. Broechoven and Parijs [3] re-evaluated this question employing the more specific calorimetric method of Novak [15] and found that plasma FFA concentration remained unchanged for at least 17 days when stored at - 20” C. The results of the present study confirm and extend this finding but indicate that FFA levels in plasma samples obtained using heparin as anticoagulant do not remain stable during long-term storage, even in the frozen state. Trichopoulou et al [5] used heparin as anticoagulant and reported significant increases in plasma FFA concentration during storage at + 5 and - 27 o C after only 24, 48 and 96 h. Such findings could explain the extremely high plasma FFA values reported by some authors [7,8] who used heparin as anticoagulant in their experiments, and can resolve the controversy [3,5,6,13] concerning the effect of different storage conditions on plasma FFA concentration.
318
Acknowledgement The author
is supported
by a grant
from the Health
Promotion
Research
Trust.
References 1 Forbes AL. Camlin JA. Effects of storage on serum non-esterified fatty acid concentrations. Proc Sot Exptl Biol Med 1959:102:709-710. 2 Overbeek GA. Fat splitting enzymes in blood. Clin Chim Acta 1957;2:1-8. 3 Broechoven CH, Parijs J. Effect of storage on plasma non-esterified fatty acid concentration. Clin Chim Acta 1968;20:530. 4 Braun JS. Freie Fettsauren im serum. Z Khn Chem Klin Biochem 1971;9:387-390. 5 Trichopoulou A, Kalaidzidou C. Kalandidi A. The relationship between plasma non-esterified fatty acid concentration and conditions of storage. Clin Chim Acta 1976:69:355-356. microdetermination of free fatty acids in plasma using 6 Degen AJM, Van der Vies. J. Enzymatic paraoxon to prevent lipolysis. Stand J Clin Lab Invest 1985;45:283-285. 7 Rognum P, Vaage 0, Hostmark A. Opstand PK. Metabolic responses to bicycle exercise after several days of physical work and energy deficiency. Stand J Clin Lab Invest 1981;41:565-571. 8 Loy SF. Conlee RK, Winder WW. Nelson AG, Amall DA, Fisher AG. Effects of 24-hour fast on cycling endurance time at two different intensities. J Appl Physiol 1986:61:654-659. DS, Gordon RS. Transport of fatty acids. Physiol Rev 1958:38:585-630. 9 Fredrickson for the medical sciences. London: Wiley, 1983. 10 Newsholme EA. Leech AR. Biochemistry micro-determination of free fatty acids in serum. 11 Noma A, Okabe H, Kita M. A new calorimetric Chn Chim Acta 1973:43:317-320. RJ. A simple enzymatic fluorimetric method for the determinatin of tri12 Gleeson M. Maughan glycerides in 10 pl of serum. Clin Chim Acta 1986;156:97-104. fatty acid in human blood plasma. II. The transport 13 Gordon RS, Cherkes A. Gates H. Unesterified function of unesterified fatty acid. J Clin Invest 1957;36:810-815. fatty acids in plasma and the metabolism of glucose. J 14 Dole VP. A relation between non-esterified Clin Invest 1956;35:150-154. ultramicro method for the determination of free fatty acids. J Lipid Res 15 Novak M. Calorimetric 1965;6:431-433.
315
CCA 03994
Short communication
Effect of heparin and storage on human plasma free fatty acid concentration Michael Gleeson Department of Environmental and Occupational Medicine, University Medical School, Aberdeen (UK)
(Received 28 January 1987; revision received 19 June 1987: accepted after revision 3 August 1987) Key words: Plasma; Free fatty acid: Heparin
Introduction In plasma and serum samples obtained from man and other mammals, spontaneous hydrolysis of esterified fatty acids into free fatty acids (FFA) has been reported [l-6]. Several investigators have described the effects of different storage conditions on plasma FFA concentration [1,3-61. This in vitro lipolysis may be of enzymatic or of physico-chemical origin [4] but it is reasonable to postulate that such lipolysis is in part due to the action of lipoprotein lipase [1,2]. Since heparin is known to activate lipoprotein lipase [2] the present study was undertaken to investigate the effect of using heparin as an anticoagulant on the concentration of FFA in human plasma stored at different temperatures. This study was prompted by the recent appearance of two papers in the literature [7,8] in which the investigators used heparin as anticoagulant for human blood and whose reported values of plasma FFA concentrations during exercise were very much higher than those described elsewhere; indeed, their values appear to be close to or above the upper concentration limit which is probably set by the plasma albumin-binding capacity [9,10]. Methods Two series of experiments were conducted. In the first, blood was obtained by venepuncture from 6 healthy adult men at rest after an overnight fast. The blood samples were collected in a dry syringe and portions were added to tubes containing Lithium Heparinate (20 U/ml blood), Potassium EDTA (1.5 mg/ml blood) or no anticoagulant. The blood in the tubes without anticoagulant was allowed to clot for 30 min and the sera separated by centrifugation (2 500 x g for 10 min at 20’ C).
Correspondence to: Dr. Michael Gleeson, Department of Environmental University Medical School, Foresterhill, Aberdeen AB9 2ZD, UK.
and Occupational Medicine,
0009-8981/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
316
From the other tubes, plasma was obtained without delay by centrifugation (2 500 x g for 10 min at 20 o C). FFA [ll] and triglyceride [12] concentrations were determined in all samples just after collection. Triplicate portions of the same plasma or serum were stored at 37, 20, 4 and -20°C and were subsequently analysed for FFA [ll] up to 36 days after collection. In the second series of experiments, serum was obtained from 4 healthy adult males as described above. The serum samples were pooled and immediately analysed for FFA [ll]. The pooled sera were divided into 1.0 ml portions, to which 50 ~1 of saline (control) or solutions of different concentrations of sodium heparinate in saline (155 mmol/l) were added to give final heparin concentrations of 0, 0.5, 2, 10, 20, 50, 100, 200 and 500 U/ml serum. The sera were incubated for 24 h at 37” and the FFA determined [ll]. Results and discussion
The results of the experiments conducted are shown in Figs. 1 and 2. FFA concentration in the samples increased with storage time and increasing the storage temperature augmented these rises (Fig. 1). At all temperatures the increase in plasma FFA was greater when heparin was used as anticoagulant compared with EDTA or no anticoagulant. After 36 days storage at - 20 o C, FFA concentrations were unchanged (compared with initial values) in serum and in plasma anticoagu-
1.0
-
0.8
-
0.6
-
4 IA. IJ.
0.4
-
E ii =
0.2
-
= 3 5E
o0
2
4
6
8
10
12
”
36
Days
Fig. 1. Influence of storage conditions on free fatty acid (FFA) concentration of human plasma using KsEDTA (open symbols) or lithium-heparin (closed symbols) as anticoagulant. Plasma was stored at + 37 o C (v, v), + 20 ’ C (0, l), + 4O C (0, W) and - 20 o C (A, A). Each point represents mean values of triplicate plasma samples from six resting postabsorptive men. FFA values for serum (not shown here) were not significantly different from those obtained with plasma containing KsEDTA. The initial FFA concentration (mean* SD) of the samples was 0.21 kO.07 (range 0.12-0.33) mmol/l and the initial triglyceride
concentration
was 0.83 f 0.25 (range 0.60-1.25)
mmol/l.
317
I
I
0
100
I 200
I
300
Heparin concentration
I
400 (U/ml
l 500 serum
)
Fig. 2. Influence of heparin concentration on the change in human serum free fatty acid (FFA) concentration after 24 h incubation at + 37’ C. Each point represents the mean of triplicate measurements at each heparin concentration. The initial FFA concentration of the pooled sera was 0.26 mmol/l.
lated with EDTA. However, in plasma containing heparin, FFA were 60% higher than initial values (Fig. 1) Figure 2 demonstrates that a wide range of heparin concentrations (OS-500 U/ml) increases the lipolytic activity of serum. Different opinions have been expressed concerning low-temperature storage of plasma or serum samples for subsequent analysis of FFA. Gordon et al [13] used oxalate as anticoagulant and showed that FFA levels do not change when plasma is stored at -20°C. In an earlier study, Forbes and Camlin [l] reported a steadily increasing FFA concentration in serum stored at -20” C. However, in the latter study FFA were analysed by the method of Dole 1141and most of the rise found by Forbes and Camlin [l] may be attributed to titration of non-FFA organic acids originating during storage [3]. Broechoven and Parijs [3] re-evaluated this question employing the more specific calorimetric method of Novak [15] and found that plasma FFA concentration remained unchanged for at least 17 days when stored at - 20” C. The results of the present study confirm and extend this finding but indicate that FFA levels in plasma samples obtained using heparin as anticoagulant do not remain stable during long-term storage, even in the frozen state. Trichopoulou et al [5] used heparin as anticoagulant and reported significant increases in plasma FFA concentration during storage at + 5 and - 27 o C after only 24, 48 and 96 h. Such findings could explain the extremely high plasma FFA values reported by some authors [7,8] who used heparin as anticoagulant in their experiments, and can resolve the controversy [3,5,6,13] concerning the effect of different storage conditions on plasma FFA concentration.
318
Acknowledgement The author
is supported
by a grant
from the Health
Promotion
Research
Trust.
References 1 Forbes AL. Camlin JA. Effects of storage on serum non-esterified fatty acid concentrations. Proc Sot Exptl Biol Med 1959:102:709-710. 2 Overbeek GA. Fat splitting enzymes in blood. Clin Chim Acta 1957;2:1-8. 3 Broechoven CH, Parijs J. Effect of storage on plasma non-esterified fatty acid concentration. Clin Chim Acta 1968;20:530. 4 Braun JS. Freie Fettsauren im serum. Z Khn Chem Klin Biochem 1971;9:387-390. 5 Trichopoulou A, Kalaidzidou C. Kalandidi A. The relationship between plasma non-esterified fatty acid concentration and conditions of storage. Clin Chim Acta 1976:69:355-356. microdetermination of free fatty acids in plasma using 6 Degen AJM, Van der Vies. J. Enzymatic paraoxon to prevent lipolysis. Stand J Clin Lab Invest 1985;45:283-285. 7 Rognum P, Vaage 0, Hostmark A. Opstand PK. Metabolic responses to bicycle exercise after several days of physical work and energy deficiency. Stand J Clin Lab Invest 1981;41:565-571. 8 Loy SF. Conlee RK, Winder WW. Nelson AG, Amall DA, Fisher AG. Effects of 24-hour fast on cycling endurance time at two different intensities. J Appl Physiol 1986:61:654-659. DS, Gordon RS. Transport of fatty acids. Physiol Rev 1958:38:585-630. 9 Fredrickson for the medical sciences. London: Wiley, 1983. 10 Newsholme EA. Leech AR. Biochemistry micro-determination of free fatty acids in serum. 11 Noma A, Okabe H, Kita M. A new calorimetric Chn Chim Acta 1973:43:317-320. RJ. A simple enzymatic fluorimetric method for the determinatin of tri12 Gleeson M. Maughan glycerides in 10 pl of serum. Clin Chim Acta 1986;156:97-104. fatty acid in human blood plasma. II. The transport 13 Gordon RS, Cherkes A. Gates H. Unesterified function of unesterified fatty acid. J Clin Invest 1957;36:810-815. fatty acids in plasma and the metabolism of glucose. J 14 Dole VP. A relation between non-esterified Clin Invest 1956;35:150-154. ultramicro method for the determination of free fatty acids. J Lipid Res 15 Novak M. Calorimetric 1965;6:431-433.