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Erythrocyte malondialdehyde release in vitro: A functional measure of vitamin E status
Clinica Chimica Actu, 151 (1985) 169-176 Elsevier
169
CCA 03271
Erythrocyte malondialdehyde release in vitro: a functional measure of vitamin E status Harry A. Cynamon
*, J. Nevin Isenberg
** and Co H. Nguyen
Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77550 (Received
February
-2776 (USA)
27th, 1985; revision May 29th. 1985)
Key words: Vitamin E; Malondialdehyde; Lipidperoxidation;
Etythrocyte
Summary
The definition for a sufficient vitamin E level has often been based on population studies that established the normal range of values for fasting plasma or serum vitamin E and more recently for vitamin E to total lipid ratios. These endpoints for vitamin E replacement strategies may not be readily achievable, particularly in the cholestatic patient for whom it is often impossible to reach and sustain normal levels even with massive doses of vitamin E. Vitamin E is believed to function as an antioxidant in vivo protecting membranes from lipid peroxidation. Malondialdehyde (MDA), a product of polyunsaturated fat peroxidation, was measured as the thiobarbiturate derivative in the supernatant following incubation of erythrocytes in hydrogen peroxide. The two different incubation conditions described here and the subsequent measurement of MDA appear to provide a sensitive functional assessment of vitamin E status. The clinical utility of this assay, which requires just 1.5 to 2.0 ml of whole blood, was demonstrated by comparing the percent of total MDA released from individuals regarded as vitamin E sufficient by conventional methods with vitamin E deficient subjects. The release of MDA from erythrocytes from vitamin E deficient subjects was clearly greater (44.1 k 18.8% vs 2.0 + 1.8%) than for control subjects ( p < 0.001).
Introduction
Aggressive, early treatment of vitamin E deficiency has become widely promoted since neuromuscular disease in chronically vitamin E deficient individuals has been * Dr. Cynamon is an American Liver Foundation ** To whom correspondence should be addressed.
0009-8981/85/$03.30
Fellow.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
170
recognized {1,2]. Attempts at replenishment of vitamin E in cholestatic individuals are hampered by uncertainties as to what constitutes a sufficient vitamin E level. Current clinical assessment means include quantitation of fasting plasma or serum vitamin E, vitamin E/total lipid ratio and the hydrogen peroxide hemolysis test. Normal vitamin E levels determined by population studies in adults range from S-20 pg/ml [3,4], Horwitt suggested that the vitamin E/total lipid ratio in healthy adults was at least 0.8 mg/g 151. Reference values for the pediatric age group are based on fewer subjects. Farrell proposed that the lower vitamin E limit in children was 3 pg/ml and that a vitamin E/total lipid ratio of 0.6 mg/g was the lower limit of the normal range [6]. Vitamin E is believed to function as an antioxidant thereby preventing membrane lipid peroxidation in vivo. Membrane polyunsaturated fatty acids, when exposed to oxidant stress, form malondialdehyde (MDA) and hydrocarbons [7]. We employed the t~obarbituri~ acid method for measurement of MDA following incubation of erythrocytes stressed by hydrogen peroxide in vitro [S]. The conditions for erythrocyte incubation utilized by prior workers (which included the addition of the erythrocyte catalase inhibitor, sodium azide) lacked the sensitivity necessary to clearly distinguish vitamin E sufficient from deficient individuals. Therefore this study was designed to clarify the conditions under which the quantitation of MDA release from erythrocytes incubated in vitro could provide a sensitive measure of vitamin E sufficiency. Furthermore, after determination of those conditions, preliminary results are presented demonstrating the potential clinical importance of this assay. Materials and methods
Reagents All reagents were of analytical grade. Trichloroacetic acid, sodium azide and hydrogen peroxide were purchased from Fisher Scientific Company, Fairlawn, NJ, USA. T~ob~bitu~c acid, sodium m-arsenite and 1,1,3,3-tetraethox~ropane were purchased from Sigma Chemical Company, St. Louis, MO, USA. Equipment A Beckman DU/Gilford recording spectrophotometer fitted with cuvettes of 1 cm light path was used for absorption measurements. The quantitation of plasma vitamin E and triglyceride was done with a Perkin-Elmer Model 203 fluorescent spectrophotometer. Statistical analyses by the two-tailed Student’s t test were performed using the CLINFO software package available at the General Clinical Research Center. Subjects Peripheral venous blood was withdrawn from all subjects after an eight to twelve hour fast. Written consent was obtained from all patients or legal guardians in the case of minors with approval of the University of Texas Medical Branch Institutional Review Board. The vitamin E status of ail subjects studied was categorized
171
using the age group criteria noted in the introduction. Three groups of subjects were evaluated. Group One consisted of noncholestatic vitamin E sufficient children ages 4 months to 10 years (n = 11). Group Two was composed of noncholestatic vitamin E sufficient adolescents and adults ages 16-61 years (n = 8). Group Three consisted of vitamin E deficient children ages 2 months to 9 years (n = 15). All but three subjects in Group Three were children with extrahepatic biliary atresia. The remaining children had neonatal hepatitis, cystic fibrosis and abetalipoproteinemia. Measurement of malondialdehyde release
Fresh whole blood (1.5 to 2.0 ml) placed into sodium citrate was immediately centrifuged at 2070 X g for 10 min. Two aliquots of 0.22 ml of packed erythrocytes were then washed once with 9.0 ml of 4°C isotonic phosphate buffered saline, pH 7.4 [9]. Next, one aliquot of the erythrocytes was resuspended to 5% (19 vol of phosphate buffered saline to 1 vol of packed RBCs) with the same phosphate buffered saline. One vol of the second aliquot of packed RBCs was resuspended in 19 vol phosphate buffer to which sodium azide (26.0 mg NaN,/lOO ml buffer) had been added. The 5% (v/v) erythrocyte suspensions were prepared for duplicate incubations under the conditions described below. Equal volumes (1.0 ml) of freshly prepared hydrogen peroxide were mixed with both suspensions just prior to incubation at 37°C in a shaking water bath. The concentration of hydrogen peroxide was determined using the extinction coefficient measured at 230 nm. Pilot studies described below demonstrated the optimal concentration of the added peroxide solution to be 0.75% for the azide suspended cells and 3.0% for those suspensions without azide. Specimen blanks with no peroxide and reagent blanks were incubated for 1 h with the test samples. One ml of trichloroacetic acid (TCA) in sodium arsenite (28 g trichloroacetic acid per dl 0.1 mol/l sodium arsenite) was added to all tubes following incubation. Arsenite has been shown to stabilize the MDA chromogen [8] and the TCA aids in precipitation of potentially interfering proteins. One-half ml of 1% thiobarbituric acid (1 g/dl) in 0.05 mol/l sodium hydroxide was added to 2.0 ml of supernatant removed from the tubes following centrifugation (2070 x g, 10 min). The specimens were then boiled in 16 X 125 mm tubes for 15 min in a water bath, cooled to room temperature and absorbance at 535 nm determined. A standard absorption curve for malondialdehyde was prepared using 1,1,3,3-tetraethoxypropane. Stock standard (400 nmol/ml) stored at 4°C was diluted with deionized water to make working standards of 1.0, 6.0, 12.0 and 20.0 nmol/ml concentrations. Selection of hydrogen peroxide concentration
In order to determine the hydrogen peroxide concentrations that yielded maximal release of MDA the following experiments were performed. For erythrocytes incubated with and without sodium azide, hydrogen peroxide was added in the following concentrations (v/v): 0.075%, 0.75%, 1.5% and 3.0% (3.0% = 880 mmol/l). This was undertaken in three vitamin E sufficient subjects from Group One and four vitamin E deficient subjects from Group Three. (See Results and Figs. 1 and 2).
172
Analytic methods for plasma lipids Vitamin E was measured by the fluorescent method of Hansen and Warwick [lo]. Total cholesterol was measured by an established spectrophotometric method [ll]. Phospholipid was quantitated spectrophotometrically as the thiocyanate complex [12]. Triglyceride was measured fluorometrically following condensation of formaldehyde to the compound 3,5-diacetyl-1,4_dihydrolutidine [13,14]. All analyses were performed in duplicate. Total lipids were calculated by summation of the individual plasma lipids. Results
Determination of incubation conditions The results of incubations with and without sodium azide at hydrogen peroxide concentrations (0.0752, 0.75%, 1.596, 3.0%) are presented for three vitamin E sufficient adults (Fig. 1). For incubations with added azide, maximal MDA release was observed with a hydrogen peroxide concentration of 0.75%. Azide was added to inhibit erythrocyte catalase which may result in the destruction of hydrogen peroxide. For erythrocyte incubations without sodium azide, essentially no release of MDA was observed at all hydrogen peroxide concentrations evaluated. We then
Vitamin
2
E
- Sufficient
Sublects
Vitamin
E
- Defioent
Subpcts
200 .-
g
__o-__-___..Y loo
---_____---
-.
1: 075
.75
1.5
20
2.5
30
,075
75
%H202
15
20
25
3.0
36 f-4202
Fig. 1. Solid lines ( ) represent incubations with sodium azide (n = 3). Broken line (- - -) represents incubations without sodium aside (n = 3). A, 0, 0 are individual subjects. + is the mean value for the three incubations done without azide all of which had minimal release of MDA as suggested by the single line shown. Fig. 2. Solid lines ( tions without sodium
), incubations with sodium azide (n = 4). Broken azide (n = 3). 0, 0, A, 0 are individual subjects.
lines (-
-
-),
incuba-
173
evaluated 4 vitamin E deficient children in the same manner (Fig. 2). For incubations with catalase inhibition nmoles of MDA released appeared similar to the control subjects. However for incubations without sodium azide (no catalase inhibition) levels of MDA released were clearly greater than those observed with controls at all hydrogen peroxide concentrations tested. For all subsequent evaluations we chose 0.75% hydrogen peroxide for incubations with sodium azide and 3.0% hydrogen peroxide for those without. This second concentration is similar to the concentration used in the standard hydrogen peroxide hemolysis test [15]. Results are subsequently presented as the percent maximal MDA release according to the following equation: MDA release (3% H 2O, ) MDA release (0.75% H,O,
plus azide)
x 100 =
%Maximal
release (%MDA)
Precision of method Ten aliquots of erythrocytes from a vitamin E sufficient adult were analyzed for MDA release with added azide (0.75% H,O,). Mean MDA release k SD was 348.1 nmol/ml RBC f 16.3 (coefficient of variation (CV) = 4.7%). Similarly ten aliquots of erythrocytes from the same vitamin E sufficient adult were analyzed for MDA release without azide (3.0% H,O,) and all ten specimens were found to have no detectable MDA release. Additionally ten aliquots of erythrocytes from a vitamin E deficient two-year-old were analyzed for MDA release without azide (3.0% H20,). Mean MDA release was 197.1 nmol/ml RBC + 9.4 (CV = 4.8%). Group comparisons Table I summarizes the plasma E, E to total fasting plasma lipid ratios and %MDA release values for the three study groups. Using the criteria noted in the introduction Groups One and Two have clearly normal vitamin E parameters. Similarly the Group Three means for vitamin E level (1.2 pg/ml) and for E/TL ratio (0.2 mg/g) are clearly in the deficient range. This marked difference in vitamin E parameters between Groups One/Two and Three are reflected by markedly different SMDA release. The vitamin E deficient group showed a SMDA release of 44.1 f 18.8% (mean f SD) in contrast to Group One (2.0 f 1.8%) and Group Two (2.1 * 1.4%) (p < 0.001).
TABLE Group
I comparisons
Group
E (w/ml)
E/TL
1 (II =11) 2(n=8) 3(n-15)
9.8 f 2.9 12.8 f 4.2 1.2* 1.9
2.5 + 0.8 2.4 f 0.4 0.2 f 0.2
Values are mean f SD; E, plasma l p -C 0.001 vs Group 1 or 2.
l
vitamin
E; E/TL,
%MDA
(mg/g)
vitamin
2.0* 1.8 2.1 f 1.4 44.1 f 18.8 *
l
E/total
plasma
lipids.
0
Oo
0
0,
10
Vltamm Fig. 3. 0
Ooo
0 OQ_o
5
are individual
0
_
_
20
15
E (&ml)
subjects.
80 0
70 60
0 0
%
z
50 i
al :
40
g
30
00 ooo
1
Vitamin E/Total
2
3
Liplds (mg/g)
Fig. 4. 0 are individual subjects
Figure 3 summarizes the relationship in all subjects studied between plasma vitamin E levels and %&MDA release. Clearly the subjects with normal vitamin E levels have essentially no fractional MDA release (SMDA < 5). Patients with very low vitamin E levels possess MDA release values in the 50% range. Finally patients with intermediate vitamin E levels have %MDA in the lo-17% range. This same relationship can also be seen to hold true when comparing the vitamin E/total lipid ratio with %MDA release (Fig. 4).
Discussion In the present study we have demonstrated that MDA release from erythrocytes incubated in vitro can provide a sensitive, functional measure of vitamin E sufficiency. Vitamin E is believed to function as an antioxidant preventing lipid
175
peroxidation in vivo. Since membranes are intrinsically rich in polyunsaturated fatty acids they are therefore susceptible to in vivo oxidation. Presumably the normal vitamin E sufficient individual is for the most part protected from in vivo oxidative damage under normal physiologic conditions. Investigators have demonstrated, however, that malondialdehyde release can be detected from normal erythrocytes incubated in vitro when exposed to peroxidant stress [8]. These incubations were carried out in the presence of sodium azide, an erythrocyte catalase inhibitor, as it prevented the destruction of hydrogen peroxide and was felt to improve reproducibility of the assay [8]. We employed similar conditions for the erythrocyte incubations with sodium azide and found MDA release quite similar for clearly vitamin E sufficient subjects and for markedly vitamin E deficient subjects. In order to increase the sensitivity of the assay we carried out a second set of incubations without catalase inhibition and found that erythrocytes from normal vitamin E sufficient subjects exhibit minimal to no detectable release of MDA (range 0 to 15.8 nmol/ml of RBCs). In contrast, erythrocytes from vitamin E deficient subjects incubated without catalase inhibition exhibited release from 40-252 nmol/ml RBCs. At first glance, one might argue that incubations without catalase inhibition should be adequate to differentiate vitamin E sufficient from deficient individuals. However values for MDA release in vitamin E sufficient subjects with catalase inhibition does vary substantially from individual to individual (range: 270-432 nmol/ml RBCs). Furthermore, since catalase is inhibited in the incubation with azide the measurement of MDA release under this set of conditions may be more a measure of the amount of polyunsaturated fatty acids present in the red cell membrane rather than a measure of vitamin E sufficiency. We view the MDA release with catalase inhibition as the maximal release of MDA possible and the MDA release without inhibition as a reflection of the erythrocyte membrane antioxidant protection. Therefore we express the data as a fractional or (A,maximal release as shown in the equation (see Results). The limitations of the use of thiobarbituric acid for the measurement of MDA have been extensively discussed elsewhere [16]. Others have referred to the use of the thiobarbituric acid method as measuring thiobarbituric reactive substances rather than malondialdehyde alone. If other minor products of lipid peroxidation do interfere with the measurement of MDA they apparently do not affect the usefulness of this assay. Under the conditions described in this paper we believe that SMDA release can be a clinically useful test for the assessment of vitamin E status that could be readily performed in clinical laboratories. The assay requires only 1.5 to 2.0 ml of whole blood, can provide results in approximately two hours following venipuncture, requires no specialized laboratory equipment and measures a biologic function of vitamin E. This assay may have particular value in monitoring the cholestatic patient during attempts to treat the vitamin E deficiency prevalent in these patients. Furthermore, it may be helpful in other patient groups such as premature infants where studies with vitamin E supplementation have not addressed the issue of whether these infants have protective vitamin E levels.
176
Acknowledgements The authors wish to thank the nursing staff of the Clinical Research Center for their excellent patient care. This study was supported in part by a grant (RR-73) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. References 1 Rosenblum JL, Keating JP, Prensky AL, Nelson JS. A progressive neurologic syndrome in children with chronic liver disease. N Engl J Med 1981; 304: 503-508. 2 Guggenheim MA. Ringel SP. Silverman A. Grabert BE. Progressive neuromuscular disease in children with chronic cholestasis and vitamin E deficiency: diagnosis and treatment with alpha tocopherols. J Pediatr 1982; 100: 51-58. 3 Bieri JG. Teets L, Belavady B, Andres EL. Serum vitamin E levels in a normal adult population in the Washington, D.C. area. Proc Sot Exptl Bio Med 1964; 117: 131-133. 4 Harris PL, Hardenbrook EG, Dean FP, Cusack ER, Jensen JL. Blood tocopherol values in normal human adults and incidence of vitamin E deficiency. Proc Sot Exptl Bio Med 1961; 107: 381-384. 5 Horwitt MK, Harvey CC. Dahm Jr CH, Searcy MT. Relationship between tocopherol and serum lipid levels for determination of nutritional adequacy. Ann NY Acad Sci 1972; 203: 223-236. 6 Farrell PM. Levin SL, Murphy D, Adams AJ. Plasma tocopherol levels and tocopherol lipid relationships in a normal population of children as compared to healthy adults. Am J Clin Nutr 1978; 31: 1720-1726. 7 Hochstein P, Rice-Evans C. Lipid peroxidation and membrane alterations in erythrocyte survival. In: Yagi K. ed. Lipid peroxides in biology and medicine. New York: Academic Press. 1982: 86. 8 Stocks J, Dormandy TL. The autoxidation of human red cell lipids induced by hydrogen peroxide. British J Haematology 1971; 20: 95-111. 9 Parpart AK, Lorenz PB. Pat-part ER. Gregg JR, Chase AM. The osmotic resistance (fragility) of human red cells. J Clin Invest 1947; 26: 636-640. 10 Hansen LG. Warwick WJ. A fluorometric micromethod for serum vitamin A and E. Am J Clin Path 1969; 51: 538-541. 11 Leffler HH, McDougald CH. Estimation of cholesterol in serum. Am J Clin Path 1963; 39: 311-315. 12 Stewart JM. Calorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 1980; 104: 10-14. 13 Belman S. The fluorometric determination of formaldehyde. Anal Chim Acta 1963; 29: 120-125. 14 Knobelsdorff A. Fluorescence clinical chemistry procedures manual. Perkin-Elmer. 1971. 15 Gordon HH. Nitowsky HM, Cornblath M. Studies of tocopherol deficiency in infants and children. Am J Dis Child 1955; 90: 669-681. 16 Yagi K. Assay for serum lipid peroxide level and its clinical significance. In: Yagi K, ed. Lipid peroxides in biology and medicine. New York: Academic Press, 1982: 223-231.
169
CCA 03271
Erythrocyte malondialdehyde release in vitro: a functional measure of vitamin E status Harry A. Cynamon
*, J. Nevin Isenberg
** and Co H. Nguyen
Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77550 (Received
February
-2776 (USA)
27th, 1985; revision May 29th. 1985)
Key words: Vitamin E; Malondialdehyde; Lipidperoxidation;
Etythrocyte
Summary
The definition for a sufficient vitamin E level has often been based on population studies that established the normal range of values for fasting plasma or serum vitamin E and more recently for vitamin E to total lipid ratios. These endpoints for vitamin E replacement strategies may not be readily achievable, particularly in the cholestatic patient for whom it is often impossible to reach and sustain normal levels even with massive doses of vitamin E. Vitamin E is believed to function as an antioxidant in vivo protecting membranes from lipid peroxidation. Malondialdehyde (MDA), a product of polyunsaturated fat peroxidation, was measured as the thiobarbiturate derivative in the supernatant following incubation of erythrocytes in hydrogen peroxide. The two different incubation conditions described here and the subsequent measurement of MDA appear to provide a sensitive functional assessment of vitamin E status. The clinical utility of this assay, which requires just 1.5 to 2.0 ml of whole blood, was demonstrated by comparing the percent of total MDA released from individuals regarded as vitamin E sufficient by conventional methods with vitamin E deficient subjects. The release of MDA from erythrocytes from vitamin E deficient subjects was clearly greater (44.1 k 18.8% vs 2.0 + 1.8%) than for control subjects ( p < 0.001).
Introduction
Aggressive, early treatment of vitamin E deficiency has become widely promoted since neuromuscular disease in chronically vitamin E deficient individuals has been * Dr. Cynamon is an American Liver Foundation ** To whom correspondence should be addressed.
0009-8981/85/$03.30
Fellow.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
170
recognized {1,2]. Attempts at replenishment of vitamin E in cholestatic individuals are hampered by uncertainties as to what constitutes a sufficient vitamin E level. Current clinical assessment means include quantitation of fasting plasma or serum vitamin E, vitamin E/total lipid ratio and the hydrogen peroxide hemolysis test. Normal vitamin E levels determined by population studies in adults range from S-20 pg/ml [3,4], Horwitt suggested that the vitamin E/total lipid ratio in healthy adults was at least 0.8 mg/g 151. Reference values for the pediatric age group are based on fewer subjects. Farrell proposed that the lower vitamin E limit in children was 3 pg/ml and that a vitamin E/total lipid ratio of 0.6 mg/g was the lower limit of the normal range [6]. Vitamin E is believed to function as an antioxidant thereby preventing membrane lipid peroxidation in vivo. Membrane polyunsaturated fatty acids, when exposed to oxidant stress, form malondialdehyde (MDA) and hydrocarbons [7]. We employed the t~obarbituri~ acid method for measurement of MDA following incubation of erythrocytes stressed by hydrogen peroxide in vitro [S]. The conditions for erythrocyte incubation utilized by prior workers (which included the addition of the erythrocyte catalase inhibitor, sodium azide) lacked the sensitivity necessary to clearly distinguish vitamin E sufficient from deficient individuals. Therefore this study was designed to clarify the conditions under which the quantitation of MDA release from erythrocytes incubated in vitro could provide a sensitive measure of vitamin E sufficiency. Furthermore, after determination of those conditions, preliminary results are presented demonstrating the potential clinical importance of this assay. Materials and methods
Reagents All reagents were of analytical grade. Trichloroacetic acid, sodium azide and hydrogen peroxide were purchased from Fisher Scientific Company, Fairlawn, NJ, USA. T~ob~bitu~c acid, sodium m-arsenite and 1,1,3,3-tetraethox~ropane were purchased from Sigma Chemical Company, St. Louis, MO, USA. Equipment A Beckman DU/Gilford recording spectrophotometer fitted with cuvettes of 1 cm light path was used for absorption measurements. The quantitation of plasma vitamin E and triglyceride was done with a Perkin-Elmer Model 203 fluorescent spectrophotometer. Statistical analyses by the two-tailed Student’s t test were performed using the CLINFO software package available at the General Clinical Research Center. Subjects Peripheral venous blood was withdrawn from all subjects after an eight to twelve hour fast. Written consent was obtained from all patients or legal guardians in the case of minors with approval of the University of Texas Medical Branch Institutional Review Board. The vitamin E status of ail subjects studied was categorized
171
using the age group criteria noted in the introduction. Three groups of subjects were evaluated. Group One consisted of noncholestatic vitamin E sufficient children ages 4 months to 10 years (n = 11). Group Two was composed of noncholestatic vitamin E sufficient adolescents and adults ages 16-61 years (n = 8). Group Three consisted of vitamin E deficient children ages 2 months to 9 years (n = 15). All but three subjects in Group Three were children with extrahepatic biliary atresia. The remaining children had neonatal hepatitis, cystic fibrosis and abetalipoproteinemia. Measurement of malondialdehyde release
Fresh whole blood (1.5 to 2.0 ml) placed into sodium citrate was immediately centrifuged at 2070 X g for 10 min. Two aliquots of 0.22 ml of packed erythrocytes were then washed once with 9.0 ml of 4°C isotonic phosphate buffered saline, pH 7.4 [9]. Next, one aliquot of the erythrocytes was resuspended to 5% (19 vol of phosphate buffered saline to 1 vol of packed RBCs) with the same phosphate buffered saline. One vol of the second aliquot of packed RBCs was resuspended in 19 vol phosphate buffer to which sodium azide (26.0 mg NaN,/lOO ml buffer) had been added. The 5% (v/v) erythrocyte suspensions were prepared for duplicate incubations under the conditions described below. Equal volumes (1.0 ml) of freshly prepared hydrogen peroxide were mixed with both suspensions just prior to incubation at 37°C in a shaking water bath. The concentration of hydrogen peroxide was determined using the extinction coefficient measured at 230 nm. Pilot studies described below demonstrated the optimal concentration of the added peroxide solution to be 0.75% for the azide suspended cells and 3.0% for those suspensions without azide. Specimen blanks with no peroxide and reagent blanks were incubated for 1 h with the test samples. One ml of trichloroacetic acid (TCA) in sodium arsenite (28 g trichloroacetic acid per dl 0.1 mol/l sodium arsenite) was added to all tubes following incubation. Arsenite has been shown to stabilize the MDA chromogen [8] and the TCA aids in precipitation of potentially interfering proteins. One-half ml of 1% thiobarbituric acid (1 g/dl) in 0.05 mol/l sodium hydroxide was added to 2.0 ml of supernatant removed from the tubes following centrifugation (2070 x g, 10 min). The specimens were then boiled in 16 X 125 mm tubes for 15 min in a water bath, cooled to room temperature and absorbance at 535 nm determined. A standard absorption curve for malondialdehyde was prepared using 1,1,3,3-tetraethoxypropane. Stock standard (400 nmol/ml) stored at 4°C was diluted with deionized water to make working standards of 1.0, 6.0, 12.0 and 20.0 nmol/ml concentrations. Selection of hydrogen peroxide concentration
In order to determine the hydrogen peroxide concentrations that yielded maximal release of MDA the following experiments were performed. For erythrocytes incubated with and without sodium azide, hydrogen peroxide was added in the following concentrations (v/v): 0.075%, 0.75%, 1.5% and 3.0% (3.0% = 880 mmol/l). This was undertaken in three vitamin E sufficient subjects from Group One and four vitamin E deficient subjects from Group Three. (See Results and Figs. 1 and 2).
172
Analytic methods for plasma lipids Vitamin E was measured by the fluorescent method of Hansen and Warwick [lo]. Total cholesterol was measured by an established spectrophotometric method [ll]. Phospholipid was quantitated spectrophotometrically as the thiocyanate complex [12]. Triglyceride was measured fluorometrically following condensation of formaldehyde to the compound 3,5-diacetyl-1,4_dihydrolutidine [13,14]. All analyses were performed in duplicate. Total lipids were calculated by summation of the individual plasma lipids. Results
Determination of incubation conditions The results of incubations with and without sodium azide at hydrogen peroxide concentrations (0.0752, 0.75%, 1.596, 3.0%) are presented for three vitamin E sufficient adults (Fig. 1). For incubations with added azide, maximal MDA release was observed with a hydrogen peroxide concentration of 0.75%. Azide was added to inhibit erythrocyte catalase which may result in the destruction of hydrogen peroxide. For erythrocyte incubations without sodium azide, essentially no release of MDA was observed at all hydrogen peroxide concentrations evaluated. We then
Vitamin
2
E
- Sufficient
Sublects
Vitamin
E
- Defioent
Subpcts
200 .-
g
__o-__-___..Y loo
---_____---
-.
1: 075
.75
1.5
20
2.5
30
,075
75
%H202
15
20
25
3.0
36 f-4202
Fig. 1. Solid lines ( ) represent incubations with sodium azide (n = 3). Broken line (- - -) represents incubations without sodium aside (n = 3). A, 0, 0 are individual subjects. + is the mean value for the three incubations done without azide all of which had minimal release of MDA as suggested by the single line shown. Fig. 2. Solid lines ( tions without sodium
), incubations with sodium azide (n = 4). Broken azide (n = 3). 0, 0, A, 0 are individual subjects.
lines (-
-
-),
incuba-
173
evaluated 4 vitamin E deficient children in the same manner (Fig. 2). For incubations with catalase inhibition nmoles of MDA released appeared similar to the control subjects. However for incubations without sodium azide (no catalase inhibition) levels of MDA released were clearly greater than those observed with controls at all hydrogen peroxide concentrations tested. For all subsequent evaluations we chose 0.75% hydrogen peroxide for incubations with sodium azide and 3.0% hydrogen peroxide for those without. This second concentration is similar to the concentration used in the standard hydrogen peroxide hemolysis test [15]. Results are subsequently presented as the percent maximal MDA release according to the following equation: MDA release (3% H 2O, ) MDA release (0.75% H,O,
plus azide)
x 100 =
%Maximal
release (%MDA)
Precision of method Ten aliquots of erythrocytes from a vitamin E sufficient adult were analyzed for MDA release with added azide (0.75% H,O,). Mean MDA release k SD was 348.1 nmol/ml RBC f 16.3 (coefficient of variation (CV) = 4.7%). Similarly ten aliquots of erythrocytes from the same vitamin E sufficient adult were analyzed for MDA release without azide (3.0% H,O,) and all ten specimens were found to have no detectable MDA release. Additionally ten aliquots of erythrocytes from a vitamin E deficient two-year-old were analyzed for MDA release without azide (3.0% H20,). Mean MDA release was 197.1 nmol/ml RBC + 9.4 (CV = 4.8%). Group comparisons Table I summarizes the plasma E, E to total fasting plasma lipid ratios and %MDA release values for the three study groups. Using the criteria noted in the introduction Groups One and Two have clearly normal vitamin E parameters. Similarly the Group Three means for vitamin E level (1.2 pg/ml) and for E/TL ratio (0.2 mg/g) are clearly in the deficient range. This marked difference in vitamin E parameters between Groups One/Two and Three are reflected by markedly different SMDA release. The vitamin E deficient group showed a SMDA release of 44.1 f 18.8% (mean f SD) in contrast to Group One (2.0 f 1.8%) and Group Two (2.1 * 1.4%) (p < 0.001).
TABLE Group
I comparisons
Group
E (w/ml)
E/TL
1 (II =11) 2(n=8) 3(n-15)
9.8 f 2.9 12.8 f 4.2 1.2* 1.9
2.5 + 0.8 2.4 f 0.4 0.2 f 0.2
Values are mean f SD; E, plasma l p -C 0.001 vs Group 1 or 2.
l
vitamin
E; E/TL,
%MDA
(mg/g)
vitamin
2.0* 1.8 2.1 f 1.4 44.1 f 18.8 *
l
E/total
plasma
lipids.
0
Oo
0
0,
10
Vltamm Fig. 3. 0
Ooo
0 OQ_o
5
are individual
0
_
_
20
15
E (&ml)
subjects.
80 0
70 60
0 0
%
z
50 i
al :
40
g
30
00 ooo
1
Vitamin E/Total
2
3
Liplds (mg/g)
Fig. 4. 0 are individual subjects
Figure 3 summarizes the relationship in all subjects studied between plasma vitamin E levels and %&MDA release. Clearly the subjects with normal vitamin E levels have essentially no fractional MDA release (SMDA < 5). Patients with very low vitamin E levels possess MDA release values in the 50% range. Finally patients with intermediate vitamin E levels have %MDA in the lo-17% range. This same relationship can also be seen to hold true when comparing the vitamin E/total lipid ratio with %MDA release (Fig. 4).
Discussion In the present study we have demonstrated that MDA release from erythrocytes incubated in vitro can provide a sensitive, functional measure of vitamin E sufficiency. Vitamin E is believed to function as an antioxidant preventing lipid
175
peroxidation in vivo. Since membranes are intrinsically rich in polyunsaturated fatty acids they are therefore susceptible to in vivo oxidation. Presumably the normal vitamin E sufficient individual is for the most part protected from in vivo oxidative damage under normal physiologic conditions. Investigators have demonstrated, however, that malondialdehyde release can be detected from normal erythrocytes incubated in vitro when exposed to peroxidant stress [8]. These incubations were carried out in the presence of sodium azide, an erythrocyte catalase inhibitor, as it prevented the destruction of hydrogen peroxide and was felt to improve reproducibility of the assay [8]. We employed similar conditions for the erythrocyte incubations with sodium azide and found MDA release quite similar for clearly vitamin E sufficient subjects and for markedly vitamin E deficient subjects. In order to increase the sensitivity of the assay we carried out a second set of incubations without catalase inhibition and found that erythrocytes from normal vitamin E sufficient subjects exhibit minimal to no detectable release of MDA (range 0 to 15.8 nmol/ml of RBCs). In contrast, erythrocytes from vitamin E deficient subjects incubated without catalase inhibition exhibited release from 40-252 nmol/ml RBCs. At first glance, one might argue that incubations without catalase inhibition should be adequate to differentiate vitamin E sufficient from deficient individuals. However values for MDA release in vitamin E sufficient subjects with catalase inhibition does vary substantially from individual to individual (range: 270-432 nmol/ml RBCs). Furthermore, since catalase is inhibited in the incubation with azide the measurement of MDA release under this set of conditions may be more a measure of the amount of polyunsaturated fatty acids present in the red cell membrane rather than a measure of vitamin E sufficiency. We view the MDA release with catalase inhibition as the maximal release of MDA possible and the MDA release without inhibition as a reflection of the erythrocyte membrane antioxidant protection. Therefore we express the data as a fractional or (A,maximal release as shown in the equation (see Results). The limitations of the use of thiobarbituric acid for the measurement of MDA have been extensively discussed elsewhere [16]. Others have referred to the use of the thiobarbituric acid method as measuring thiobarbituric reactive substances rather than malondialdehyde alone. If other minor products of lipid peroxidation do interfere with the measurement of MDA they apparently do not affect the usefulness of this assay. Under the conditions described in this paper we believe that SMDA release can be a clinically useful test for the assessment of vitamin E status that could be readily performed in clinical laboratories. The assay requires only 1.5 to 2.0 ml of whole blood, can provide results in approximately two hours following venipuncture, requires no specialized laboratory equipment and measures a biologic function of vitamin E. This assay may have particular value in monitoring the cholestatic patient during attempts to treat the vitamin E deficiency prevalent in these patients. Furthermore, it may be helpful in other patient groups such as premature infants where studies with vitamin E supplementation have not addressed the issue of whether these infants have protective vitamin E levels.
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Acknowledgements The authors wish to thank the nursing staff of the Clinical Research Center for their excellent patient care. This study was supported in part by a grant (RR-73) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health. References 1 Rosenblum JL, Keating JP, Prensky AL, Nelson JS. A progressive neurologic syndrome in children with chronic liver disease. N Engl J Med 1981; 304: 503-508. 2 Guggenheim MA. Ringel SP. Silverman A. Grabert BE. Progressive neuromuscular disease in children with chronic cholestasis and vitamin E deficiency: diagnosis and treatment with alpha tocopherols. J Pediatr 1982; 100: 51-58. 3 Bieri JG. Teets L, Belavady B, Andres EL. Serum vitamin E levels in a normal adult population in the Washington, D.C. area. Proc Sot Exptl Bio Med 1964; 117: 131-133. 4 Harris PL, Hardenbrook EG, Dean FP, Cusack ER, Jensen JL. Blood tocopherol values in normal human adults and incidence of vitamin E deficiency. Proc Sot Exptl Bio Med 1961; 107: 381-384. 5 Horwitt MK, Harvey CC. Dahm Jr CH, Searcy MT. Relationship between tocopherol and serum lipid levels for determination of nutritional adequacy. Ann NY Acad Sci 1972; 203: 223-236. 6 Farrell PM. Levin SL, Murphy D, Adams AJ. Plasma tocopherol levels and tocopherol lipid relationships in a normal population of children as compared to healthy adults. Am J Clin Nutr 1978; 31: 1720-1726. 7 Hochstein P, Rice-Evans C. Lipid peroxidation and membrane alterations in erythrocyte survival. In: Yagi K. ed. Lipid peroxides in biology and medicine. New York: Academic Press. 1982: 86. 8 Stocks J, Dormandy TL. The autoxidation of human red cell lipids induced by hydrogen peroxide. British J Haematology 1971; 20: 95-111. 9 Parpart AK, Lorenz PB. Pat-part ER. Gregg JR, Chase AM. The osmotic resistance (fragility) of human red cells. J Clin Invest 1947; 26: 636-640. 10 Hansen LG. Warwick WJ. A fluorometric micromethod for serum vitamin A and E. Am J Clin Path 1969; 51: 538-541. 11 Leffler HH, McDougald CH. Estimation of cholesterol in serum. Am J Clin Path 1963; 39: 311-315. 12 Stewart JM. Calorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 1980; 104: 10-14. 13 Belman S. The fluorometric determination of formaldehyde. Anal Chim Acta 1963; 29: 120-125. 14 Knobelsdorff A. Fluorescence clinical chemistry procedures manual. Perkin-Elmer. 1971. 15 Gordon HH. Nitowsky HM, Cornblath M. Studies of tocopherol deficiency in infants and children. Am J Dis Child 1955; 90: 669-681. 16 Yagi K. Assay for serum lipid peroxide level and its clinical significance. In: Yagi K, ed. Lipid peroxides in biology and medicine. New York: Academic Press, 1982: 223-231.