Malondialdehyde formation in stored plasma

Vol, 95, No. 4,1980

BIOCHEMICAL AND.BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1663-1672

August 29, 1980

MALONDIALDEHYDE FORMATION IN STORED PLASMA

Diana M. Lee Laboratory of Lipid and Lipoprotein Studies, Oklahoma Medical Research Foundation, and the Department of Biochemistry and Molecular Biology, The University of Oklahoma, Oklahoma City, Ok. 73104 Received

June

11,1980

SU~4MARY: The formation of malondialdehyde in plasma during storage was used as an indicator of lipid peroxidation~ Malondialdehyde concentrations were measured in plasma stored at 4°C in the presence of various preservative reagents. A rapid rate of formation of malondialdehyde (15.0 + 14.0 nmol/dl/ week) was observed in control plasma preserved with diisopropylfluorophosphate and NaN 3. Addition of the antioxidant glutathione or EDTA slowed the rate to 4.8 + 3.0 nmol/dl/week or 4~0 + 3.0 nmol/dl/week respectively. The combined use--of EDTA and glutathione further reduced the rate to 1.6 + 1.6 nmol/dl/week. Therefore, the use of both glutathione and EDTA provided a 4fold improvement over EDTA alone in protection of the plasma from lipid peroxidation. Lipids in lipoproteins of all density classes contributed to the malondialdehyde formation. INTRODUCTION During the course of studying apolipoprotein B (ApoB*), the major protein moiety of low density lipoproteins

(LDL), we observed that the solubil-

ity of ApoB decreased as the age of the LDL sample increased.

The decrease

in solubility of ApoB also occurred when the LDL were extracted with ether which had not been freed of the hydrogen peroxide.

These observations have

prompted us to suspect that aging of LDL may be associated with lipid peroxidation, even though all LDL preparations contained 0.05% EDTA, a reagent which chelates the trace metals catalyzing lipid peroxidation

(i, 2).

If it

is true that lipid peroxidation occurs with aging of LDL, we wondered if lipid peroxidation occurs with aging of plasma. Lipid peroxidation is believed to proceed via free radical chain reactions

(3).

It has been reported that common proteins such as serum albumin,

e-globulin, and cytochrome C etc. when subjected to lipid peroxidation in *The abbreviations used are: ApoB, apolipoprotein B; LDL, low density lipoproteins, d=i.006-i.063 g/ml; MDA, malondialdehyde; DFP, diisopropylfluorophosphate; EDTA, ethylenediamine tetraacetate disodium salt.

0006-291X/80/161663-10501.00/0 1663

Copyr~ht Q 1980byAcademic Press, ~c. AII rights o f reproduction in any form reserved.

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

aqueous solutions, undergo major reactions of polymerization, polypeptide chain scission, chemical changes in individual amino acids and loss of solubility hyde

(4, 5).

(MDA).

One of the end products of lipid peroxidation is malondialde-

MDA is a bifunctional cross-linking agent

(6).

It may form con-

densation products with proteins and yield intra- or inter-molecular crosslinking

(7, 8).

Using sodium dodecyl sulfate-polyacrylamid e gel electropho-

resis, Janado et al (9) most recently demonstrated that MDA could cause the formation of oligomers of bovine serum albumin. MDA affects not only the molecular weights of proteins, but also their net charges.

For every mole of ~-amino group of lysine reacting with MDA,

there is a net decrease of one positive charge°

This may increase the pro-

tein's anodic electrophoretic mobility in a basic buffer, or lower its pI in an isoelectric focusing system.

It should be realized that these altered

protein forms, caused by lipid peroxidation in the starting materials or during isolation, may be mistaken for native isoproteins or polymorphs. Therefore, it is important to recognize the conditions under which MDA is formed. The purpose of this study was to use ~ A

as a marker for the assessment

of lipid peroxidation occurring in outdated plasma during storage under various conditions.

The conditions chosen for testing were those commonly used

for protection of plasma from micro-organism growth, protease degradation and lipid peroxidation.

That is, the plasma was preserved in the presence Of

NAN3, diisopropylfluorophosphate

(DFP) and EDTA.

In addition, the water sol-

u b l e a n t i o x i d a n t glutathione was incorporated for comparison.

MATERIALS AND METHODS Outdated human plasma was obtained from a local blood bank. The plasma was drawn into vacuumed plastic bags from seven apparently healthy subjects and stored at 4°C. The initial ages of the samples were purposely chosen to cover a wide range: 3 to 56 weeks. None of the plasma showed the presence of any red cells, hemolysis nor visible suspension. All plasma looked clear and indistinguishable to the naked eye as to age. Upon receipt of the plasman aliquots for lipid analyses were withdrawn from each bag with a hypodermic syringe at the upper portion of the plastic

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tubing leading to the bag. Immediately thereafter, the preservatives DFP and NaN 3 were injected into each bag to final concentrations of 1 m M DFP and 0.05% NaN 3. Air was then squeezed out, and each bag was immediately sealed. This step allowed only a minute amount of air to come in contact with the plasma. The plasma was stored in the bags at 4°C for another six weeks; then the bags were opened and the plasma exposed to air. This time was taken as zero time for MDA measurement. Four glass tubes were filled with plasma from each sample for the four different experimental conditions. Glutathione and EDTA were added to the appropriate groups so that the reagents used for the four groups were as follows: i) Control: 1 m M DFP + 0.05% NaN 3 only, 2) 1 m M DFP + 0.05% NaN 3 + 0.02% glutathione, 3) 1 m M DFP + 0.05% NaN 3 + 0.05% EDTA, and 4) 1 mM DFP + 0.05% NaN 3 + 0.02% glutathione and 0.05% EDTA. Samples were rubber-stoppered and stored at 4°C all the time except during aliquoting, which was carried out at room temperature. MDA was estimated by the thiobarbituric acid method (i0), as modified by Fong et al (ii). Triplicate aliquots of 1 ml plasma were used for each determination. The pink color of thiobarbituric acid-reacting compounds was developed by heating the mixture at 60°C for 90 minutes. This heating condition minimized the interference of sugars with thiobarbituric acid. The turbidity due to lipid was ex_tracted with chloroform. The intensity of the color in the aqueous phase was m e a s u r e d with a Gilford spectrophotometer at 532 nm. Malondialdehyde tetramethyl acetal (Eastman) was used as standard and was carried through the same steps. A linear relationship of MDA concentration versus optical density was found for concentrations from 0-5 nmol. MDA determinations on the test samples were made at 0, 2, 4, 6, 9, ii, 13, 21 and 39 weeks. Lipid analyses were performed on the whole plasma before additives were included. Triglycerides and total cholesterol were determined with an autoanalyzer (12, 13). RESULTS The plasma triglyceride are shown in Table 1. agulant

Considering

in the plasma bags,

ject D whose triglyceride

TABLE l: subjects

and total cholesterol an approximate

levels for all subjects 15% dilution due to antico-

all subjects had normal

lipid levels except sub-

level was slightly raised above normal.

PLASMA LIPID LEVELS OF SUBJECTS STUDIED

Age of plasma in vacuum bags (weeks)

Triglycerides mg/dl ~mol/ml

Total cholesterol mg/dl ~mol/ml

A

i0

74

0.84

173

4.46

B

56

154

1.74

90

2.32

C

7

55

0.62

102

2.63

D

3

190

2.15

189

4.89

E

21

129

1.45

134

3.46

F

8

80

0.90

133

3.43

G

29

I00

1.12

140

3.60

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A /

3

C

1

C 0

I

I

l

I

10

20

30

40

10

0

E

<[ c3

V

•

V

;

,Ill

I

I

I.

0 2 4 6 8 10

20

30

40

I,tlll 0 2 4 6 810

TIME (weeks) Fig.

1 A-C

1666

l

I

I

20

30

4o

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E

9'

"

v

•

E r" i

t i

i

i

i

i

i

I

I

I

20

30

40

C-~

G

w

-

i-

~ l l l J

4 6 8 10

I

I

I

20

30

40

J l l l J

4 6 8 10

TIME (weeks)

Fig. 1 A-G: MDA concentrations in plasma of subjects A through G vs storage time after plasma was exposed to air. - ~ - denotes control plasma containing 1 mM DFP + 0.05% NAN3; - Q - , plasma containing 1 m M DFP + 0.05% NaN 3 + 0.02% glutathione; - O - , plasma containing 1 mM DFP + 0.05% NaN 3 + 0.05% EDTA; and - • - , plasma containing 1 mM DFP + 0.05% NaN 3 + 0.02% glutathione + 0.05% EDTA. The experimental conditions are described in Methods.

L o w v a l u e s of M D A w e r e f o u n d in the p l a s m a of a l l s u b j e c t s at zero-time. N o t s h o w n in the figures, the a v e r a g e z e r o - t i m e v a l u e for M D A w a s 0.32 n m o l / ml w i t h a r a n g e of 0 . 2 2 - 0 . 4 0 n m o l / m l of p l a s m a .

This n a r r o w r a n g e of M D A

v a l u e s d o e s n o t r e f l e c t the w i d e r a n g e of age of the p l a s m a b e i n g s t o r e d under vacuum

(Table i), s u g g e s t i n g t h a t the f o r m a t i o n of M D A in p l a s m a u n d e r

v a c u u m is p r o b a b l y e x t r e m e l y slow if any.

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O n c e the p l a s m a w a s e x p o s e d to air, M D A f o r m e d r a p i d l y in c o n t r o l samples

(Fig~ 1 A-G) s t h o u g h the rate v a r i e d f r o m i n d i v i d u a l to i n d i v i d u a l °

G e n e r a l l y , w i t h i n the i n i t i a l I0 w e e k s , l i n e a r l y w i t h time. formation.

the c o n c e n t r a t i o n s of M D A i n c r e a s e d

The p r e s e n c e of g l u t a t h i o n e or E D T A s l o w e d d o w n the M D A

The u s e of g l u t a t h i o n e a n d E D T A in c o m b i n a t i o n s u r p a s s e d the ef-

fect of e i t h e r a l o n e

(Fig. 1 A-G).

The r e s u l t s are s u m m a r i z e d in T a b l e 2.

The a v e r a g e i n i t i a l rate of M D A f o r m a t i o n for the c o n t r o l g r o u p w a s 15.0 + 14.0 n m o l / d l / w e e k .

The p r e s e n c e of g l u t a t i o n e d e c r e a s e d the M D A f o r m a t i o n to

a n a v e r a g e of 4.8 + 3 . 0 n m o l / d l / w e e k ; the use of E D T A d e c r e a s e d the r a t e to 4.0 ~ 3.0 n m o l / d l / w e e k , and the c o m b i n a t i o n of E D T A and g l u t a t h i o n e s i g n i f i c a n t l y r e d u c e d the M D A f o r m a t i o n to 1.6 + 1.6 n m o l / d l / w e e k .

Thus,

the u s e of

b o t h E D T A a n d g l u t a t h i o n e is a b o u t 13 t i m e s m o r e e f f e c t i v e t h a n the c o n t r o l c o n d i t i o n s , and a 4 - f o l d i m p r o v e m e n t o v e r E D T A alone. In all s e v e n subjects, the c o n c e n t r a t i o n of M D A i n c r e a s e d w i t h t i m e u n der all c o n d i t i o n s

(with or w i t h o u t E D T A and g l u t a t h i o n e ) .

However,

the r a t e

of f o r m a t i o n of M D A a f t e r e x p o s u r e of the p l a s m a to air w a s n o t c o r r e l a t e d to

TABLE 2:

RATE OF MDA FORMATION

Initial rate of MDA formation Subjects

Comparison

nmol/dl/week Control

GSH

EDTA

EG

Control/EG

EDTA/EG

3.0

4.2

2.7

A

12.5

8.2

8.2

B

10.5

1.7

1.2

1.0

10.5

1.2

46.0

8.7

6.8

4.6

i0.0

1.5

D

11.3

6.2

3.9

1.5

7.5

2.6

E

13.0

4.3

5.8

0.5

26.0

11.6

F

5.0

1.8

1.8

0.3

16.7

6.0

G

7.0

2.5

0.5

0.5

14.0

1.0

4.0

1.6

C

Average

Note:

15.0

4.8

~ 14.0

.+ 3.0

.+ 3.0 .+ 1.6 .

.

12.7

3.8

+ 7.1

+ 3.8

Control=plasma containing 1 mM DFP + 0.05% NAN3; GSH=control + 0.02% glutathione; EDTA=control + 0.05% EDTA; EG=control + 0.02% glutathione + 0.05% EDTA. Averages are expressed as mean + standard deviation.

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3=

T

~..4o

,-E

.o o

~_ . 3 -

E~ ,9o

o ~ .2o

!! II

tlJ

0

~o

0

~o

T

o

4'o

~o

Age of Plasma in Vacuum (weeks)

Fig. 2: Initial rates of plasma MDA formation in various preservatives after exposure to air vs length of time of plasma stored under vacuum. - V - , plasma containing 1 m M DFP + 0.05% NAN3; , O - , plasma containing 1 m M DFP + 0.05% NaN 3 + 0.05% EDTA; and - ~ - , plasma containing 1 m M DFP + 0.05% NaN 3 + 0.05% EDTA + 0.02% glutathione. Rates under different preservatives from the same subject are connected with a line.

.5"

~.* e,-

E .2" a

0 rt"

0

0

.~

,'.o :

,s

2~.o

Prlasma Tricjlyceride (/J.mol/ml) Fig. 3: Initial rates of plasma M D A formation in various preservatives plasma triglycerides. The symbols are the same as in Fig. 2.

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the length of time the samples had been held under vacuum

(Fig.

2).

of MDA formation was not correlated

to the plasma triglyceride

3) nor to cholesterol

4) for these normal lipidemic

content

(Fig.

The rate

content

(Fig.

subjects.

DISCUSSION EDTA has been widely used as a plasma preservative. assumed that the natural protect

antioxidants

it from oxidation.

present

It is generally

in plasma are sufficient

to

This study has shown that when plasma is exposed

to air, the MDA concentration

increases

linearly with time during storage

even when EDTA is present. Although correlate

the rate of MDA formation among these seven subjects

with their normal plasma triglyceride

results do not rule out the possibility larger population iations

including hyper- or

in the rate of MDA formation

or cholesterol

did not

levels,

the

that correlations might be found in a

hypo-lipoproteinemic

subjects.

The v a ~

among subjects ~ a y be a reflection

oo.5-

T

~.4E c c

o

.3-

E .2'

'~

.I-

n.-

0

'

0

I

]

2

,

I:!I •

4

Plasma Total Cholesterol (~mol/ml) Fig. 4: Initial rates of plasma MDA formation in various preservatives vs plasma total cholesterols. The symbols are the same as in Fig. 2.

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both their plasma antioxidant contents, e.g. vitamin E and carotenoids, and their polyunsaturated fatty acid contents, particularly those containing H H H double bonds in the fashion =C-C-C=o i H

While this study was in progress, Schuh et al (14) reported that NaN 3 might promote lipid peroxidation in LDL.

For this reason we investigated

plasma stored in the absence of NAN3, with or without penicillin-G 500 units/ ml, streptomycin sulfate 50 ~g/ml, and/or thimerosal 0.05%, and 0.05% EDTA. The increase in MDA concentration upon storage of plasma persisted. To establish whether lipoproteins other than LDL provide lipids for MDA formation in plasma we tested very low density lipoproteins, high density lipoproteins and subfractions of low- and high-density lipoproteins, originally isolated in the presence of 0.05% EDTA from fresh plasma and outdated plasma (15).

All lipoprotein samples had increased MDA concentration upon storage

irrespective of their density properties and regardless of whether they were originally isolated from fresh plasma or not.

In contrast, two LDL samples

isolated from fresh plasma in the presence of EDTA, glutathione and N 2 gave negative reactions with thiobarbituric acid following storage for 14 days in N 2 at 4°C.

These results suggest that lipids in all lipoproteins are suscep-

tible to peroxidation. Lipid peroxidation affects not only the solubility and structure of plasma components but also their uptake by cells.

Fogelman et al (16) demon-

strated that the uptake of the MDA-modified LDL and the native LDL by human monocyte-macrophages were significantly different. protein and lipoprotein to be used for metabolic

We suggest that plasma or

structural studies ought

to be isolated under oxidation-free conditions.

ACKNOWLEDGEMENTS The author would like to thank Mr. W. H. Kuo and Mr, Douglas Lawrence for their capable technical assistance, and Mrs. Sherry Fischer for typing the manuscript. This work was supported in part by Grant HL-14807 and Program Project HL-23181 from the Department of Health, Education and Welfare and the resources of the Oklahoma Medical Research Foundation.

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REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13.

14. 15. 16.

Ray, B. R., Davisson, E. O., and Crespi, H° L. (1954) J. Phys. Chem. 58, 841-846. Ontko, J . A . (1970) J. Lipid Res. ii, 367-3ff5o Tappel, A. L. (1973) Federation Proceedings 32, 1870-1874. Roubal, W. T. and Tappel, A. L. (1966) Arch. Biochem. Biophys. 113, 5-8. Roubal, W. T. and Tappel, A. L. (1966) Arch. Biochem. Biophys. 113, 150155. Wold, F. (1972) in Methods in Enzymology, Hirs, C. H. W. and Timasheff, S. N. eds. 25, pp. 623-651, Academic Press, New York. Chio, K. S. and Tappel, A. L. (1969) Biochemistry 8, 2827-2832. Chio, K. S. and Tappel, A. L. (1969) Biochemistry 8, 2821-2827. Janado, M., Yano, Y., Nakamori, H. and Nishida, T. (1979) J. Biochem. 86, 177-182. Bernheim, F., Bernheim, M. L. C. and Wilbur, K . M . (1948) Biol. Chem. 174, 257-264. Fong, K.-L., McCay, P. B., Poyer, J. L., Keele, B. B. and Misra, H. (1973) J. Biol. Chem. 248, 7792-7797. Kessler, G. and Lederer, H. (1966) in Automation in Analytical Chemistry, ed. L. T. Skeggs, pp. 341-344, Mediad Inc~, New York. Rush, R. L., Leon, L. and Turrell, J. (1970) in Advances in Automated Analyses, Technicon Internati0nal Congresff, I. Clinical Analysis, pp. 503-507, Thurman Associates, Miami, FI. Schuh, J., Fairclough, Jr., G. F. and Haschemeyer, R. H. (1978) Proc. Natl. Acad. Sci. 75, 3173-3177. Alaupovic, P., Lee, D. M. and McConathy, W. J. (1972) Biochim, BiophysL Acta 260, 689-709. Fogelman, A. M., Shechter, I., Seager, J., Hokom, M., Child, J. S. and Edwards, P. A. (1980) Proc. Natl. Acad. Sci. 77, 2214-2218.

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