Oxidative stress in abetalipoproteinemia patients receiving long-term vitamin E and vitamin A supplementation1,2

Esther Granot and Ron Kohen

ABSTRACT Background: Patients with abetalipoproteinemia develop progressive ataxic neuropathy and retinopathy that are thought to be due, in part, to oxidative damage resulting from deficiencies of vitamins E and A. Objective: The goal was to determine the degree of oxidative stress in abetalipoproteinemia patients who had received vitamin E (100 mg/kg) and vitamin A (10 00015 000 IU/d) since infancy. Design: Ten patients aged 325 y were studied. Assessed were plasma carbonyl concentrations as a marker of oxidative damage to proteins; total plasma oxidizability, which was used to evaluate the susceptibility of plasma lipoproteins to oxidation; and cyclic voltammetry, which represents the overall reducing and antioxidant capacity stemming from low-molecular-weight antioxidants in plasma. Results: Concentrations of plasma carbonyls did not differ significantly between patients and control subjects (x SE: 0.5670 0.031 and 0.5039 0.0134 nmol/mg protein, respectively). The lag phase of plasma oxidizability was 28.03 3.16 min in the patients and 24.0 2.79 min in healthy subjects in whom oxidizability of isolated HDL was measured (NS). Cyclic voltammetry showed a peak potential of 330 8.3 mV in all samples studied, denoting that the same antioxidants were present in the plasma of the patients and the control subjects. The anodic current of the samples, a measure of the concentration of hydrophilic lowmolecular-weight antioxidants, was 5.227 0.25 and 5.38 0.20 in the patients and the control subjects, respectively (NS). Conclusion: Enhanced oxidative stress is not apparent in the plasma of abetalipoproteinemia patients receiving long-term supplementation with vitamins E and A. Am J Clin Nutr 2004;79: 22630. KEY WORDS min E, vitamin A Abetalipoproteinemia, oxidative stress, vita-

INTRODUCTION

Abetalipoproteinemia is a rare autosomal recessive disorder characterized by defective assembly and secretion of plasma apolipoprotein Bcontaining lipoproteins. The disorder results from mutations in the gene encoding the microsomal triacylglycerol transfer protein (1, 2). Abetalipoproteinemia patients have extremely low concentrations of plasma cholesterol and triacylglycerol and an almost complete lack of lipoproteins in the density ranges of chylomicrons, VLDLs, and LDLs. Plasma lipids are carried almost entirely in the HDL class.

Typically, patients with abetalipoproteinemia have malabsorption and may present with failure to thrive in early childhood. A progressive ataxic neuropathic disease and retinopathy that develop in later childhood are attributed, in part, to a deficiency of fat-soluble vitamins, specifically, vitamins E and A (3, 4). Both vitamins possess antioxidant properties, and vitamin E is a proven potent suppressor of lipid peroxidation (5, 6). Treatment with vitamins E and A, currently offered to abetalipoproteinemia patients from the time of diagnosis, is believed to prevent neurologic and retinal lesions, although it does not reverse the abnormalities already present (7, 8). In a previous study (9), we showed fundoscopic and functional retinal changes in 10 abetalipoproteinemia patients and 3 homozygous hypobetalipoproteinemia patients who were followed by electrophysiologic and visual field studies for 425 y (x: 14.4 y). Thus, although combined therapy with vitamins E and A does confer protection against severe retinal degeneration, the retinal changes we observed suggest ongoing oxidative damage despite antioxidant therapy. HDL is the major circulating lipoprotein in abetalipoproteinemia patients. Oxidative modification of HDL impairs biological functions critical to its role in reverse cholesterol transport (10, 11). Recent studies focused on the effect of dietary fat in the oxidative susceptibility of HDL (12, 13). In abetalipoproteinemia, the oxidation of HDL is expected to be influenced not only by the low plasma concentrations of fatsoluble antioxidant vitamins, primarily vitamin E, but also by the unique composition of the HDL particle and by the characteristic plasma fatty acid profile. HDL particles in abetalipoproteinemia patients are richer in cholesterol ester and poorer in phospholipids than are HDL particles in healthy persons and are deficient in polyunsaturated fatty acids, specifically, linoleic acid (4, 14). The present study was performed to determine markers of oxidative damage and antioxidant capacity in the plasma of abetalipoproteinemia patients receiving long-term therapy with vitamins E and A. We also aimed to further define the effect of
From the Department of Pediatrics, Hadassah University Hospital, Jerusalem (EG), and the Department of Pharmaceutics, School of Pharmacy, Hebrew University-Hadassah Medical School, Jerusalem (RK). 2 Reprints not available. Address correspondence to E Granot, Department of Pediatrics, Hadassah University Hospital, PO Box 12000, Jerusalem, Israel. E-mail: etgranot@md.huji.ac.il. Received May 13, 2003. Accepted for publication July 22, 2003.
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Am J Clin Nutr 2004;79:226 30. Printed in USA. 2004 American Society for Clinical Nutrition

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antioxidant vitamins and polyunsaturated fatty acids on HDL oxidation.

SUBJECTS AND METHODS

Patients Ten abetalipoproteinemia patients aged 325 y (x: 14.6 y) were studied. All of the patients had received a diagnosis of abetalipoproteinemia during their first year of life and had since received 100 mg vitamin E/kg and 10 00015 000 IU vitamin A/d, as previously described (8). The study was approved by the Hadassah Hospital Ethics Committee. Venous blood was drawn during a routine clinic visit into EDTA-containing evacuated tubes. Plasma was separated at 4 C by low-speed centrifugation (3000 g). An aliquot was removed for lipoprotein analysis, and the remaining plasma was kept at 70 C until analyzed. Control subjects Results of carbonyl concentrations and cyclic voltammetry were compared with reference values for 10 age-matched healthy control subjects derived from a large database available in our laboratory (15; E Granot and R Kohen, unpublished observations, 19992003). In abetalipoproteinemia, HDL is essentially the sole lipoprotein in plasma; thus, the results of plasma oxidizability in abetalipoproteinemia patients were not compared with total plasma oxidizability measurements in healthy control subjects but with those obtained for the oxidizability of HDL isolated from 6 healthy subjects. Determination of plasma carbonyl content Carbonyl concentrations in plasma were measured by the spectrophotometric assay described by Reznick and Packer (16). Briefly, to 200 L plasma, 4 mL of 10 mmol 2,4dinitrophenylhydrazine (DNPH)/L in 2 mol HCl/L was added. In another tube, 4 mL of 2 mol HCl/L was added to 200 L plasma (of the same patient). The tubes were left in the dark for 1 h at room temperature and were mixed by vortex every 15 min. Five milliliters of 20% trichloroacetic acid solution was then added to both tubes for a 10-min incubation on ice, after which the tubes were centrifuged (3000 g, 5 min, 4 C). The supernatant fluid was discarded and another wash was performed by using 4 mL 10% trichloroacetic acid. The protein pellets were broken mechanically and washed 3 times with ethanolethyl acetate to remove free DNPH and lipid contaminants. The final precipitates were dissolved in 2 mL of 6 mol guanidine hydrochloride/L, and insoluble materials were removed by additional centrifugation (3000 g, 5 min, 4 C). The carbonyl content was calculated by obtaining the spectra of the DNPH-treated samples at 355390 nm (scanned against the samples treated with 2 mol HCl/L). The carbonyl content (nmol/mL) was calculated from the peak absorbance (355390 nm) by using an absorption coefficient. The amount of protein in the sample was determined with Bradford reagent, and the carbonyl content was standardized to the protein content. Measurement of plasma oxidizability by the conjugated diene method The procedure performed, a slight modification of the assay described by Kontush and Beisiegel (17), measures the suscep-

tibility of lipoproteins to oxidation and is based on the continuous photometric detection of lipid hydroperoxides possessing a conjugated diene structure produced by exposure to oxidizing radicals. In brief, 20 L plasma was diluted with 2.98 mL phosphate-buffered saline containing 0.16 mol NaCl/L, to which 50 L 2.2-azobis(2-amidinopropane) hydrochloride was added. Another tube with 50 L 2.2-azobis(2-amidinopropane) hydrochloride diluted in 3 mL phosphate-buffered saline served as a reference solution. Samples, in cuvettes, were incubated in a spectrophotometer (Uvikon 933; Kontron AG Corp, Zurich, Switzerland) at 37 C for 160 min, during which time sample absorbance at 234 nm was measured every 12 s. With a sample of distilled water, as control, no optical density (OD) changes were observed over a 160-min period. Kinetic data were analyzed by using software supplied with the spectrophotometer. Two parameters were calculated for each sample: 1) the lag phase: the period in which no increase in absorbance at 234 nm was observed, measured from the initiation of oxidation until conjugated dienes began to accumulate, and 2) the oxidation rate of the plasma: measured during the period in which conjugated dienes accumulated and expressed as the increase in absorbance at 234 nm as a function of time (eg, the overall change in OD readings over 160 min was divided by 160 to obtain the slope of the change in OD/min). The lag phase correlates with the antioxidant properties of plasma, whereas the oxidation rate correlates with the intrinsic properties of the lipoproteins (eg, their oxidizable substrates), mainly their unsaturated fatty acid content. Cyclic voltammetry Cyclic voltammetry and its tracings represent overall reducing antioxidant capacityie, that stemming from the various low-molecular-weight antioxidant components (including ascorbic acid, uric acid, glutathione, NADH, and polyphenols) that act directly with the reactive oxygen speciesbut not the specific contribution of each compound (18). The cyclic voltammetry technique provides a highly sensitive measure of hydrophilic low-molecular-weight antioxidants. Lipophilic low-molecular-weight antioxidants, which are partially bound to plasma lipoproteins, cannot be accurately assessed by this technique without appropriate extraction procedures. The cyclic voltammetry tracings of human plasma samples record the biological peak potential, which is characteristic for the various low-molecular-weight antioxidants, and the anodic current, which indicates the total concentration of the low-molecularweight antioxidants present (18, 19). Cyclic voltammetry was carried out with a BAS model CV-1B cyclic voltammetry apparatus (Bioanalytical Systems, West Lafayette, IN). Cyclic voltammetry tracings were recorded from 0.3 to 1.3 V at a rate of 100 mV/s versus an Ag/AgCl reference electrode. A three-electrode system was used throughout the study. The working electrode was a glassy carbon disc (BAS MF-2012; Bioanalytical Systems), 3.2 mm in diameter. Platinum wire served as a counter electrode. The working electrode was polished before each measurement with a polishing kit (BAS PK-1; Bioanalytical Systems). Analysis of cyclic voltammetry tracings and determination of peak potential E1/2 and the detector anodic current ( A) were carried out as previously described (19).

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Concentrations of plasma vitamin E and -carotene Plasma concentrations of vitamin E ( -tocopherol) were measured as described by Hashim and Schuttringer (20). Plasma concentrations of -carotene, a precursor of vitamin A, were measured as described by Kaplan (21). Isolation of HDL from plasma of healthy subjects for measurement of HDL oxidizability After the subjects had fasted for 12 h, venous blood was drawn into EDTA-containing evacuated tubes, and plasma was separated at 4 C by low-speed centrifugation (3000 g). HDL was separated by salt density ultracentrifugation (4C, 48 h, 39 000 g) at densities of 1.0631.21 g/mL, and HDLcholesterol concentrations were measured by using a commercial diagnostic kit (Boehringer, Mannheim, Germany). Statistical analysis Statistical analysis for significant differences was performed with the use of Students t test and the Mann-Whitney test for unpaired data with GRAPHPAD INSTAT 3.0 (GraphPad Software Inc, San Diego). Results are presented as ranges or as means SEs.

Plasma concentrations of vitamin E and -carotene In the patients, plasma concentrations of -tocopherol ranged from 0.01 to 0.35 mg/100 mL. Concentrations in individual patients varied at different time periods within this range, with mean concentrations of 0.10.2 mg/100 mL. Reference -tocopherol concentrations ranged from 0.5 to 1.5 mg/100 mL. Plasma concentrations of -carotene ranged from 8 to 120 mg/100 mL, with mean concentrations of 2045 mg/100 mL (reference range: 20300 mg/100 mL).

DISCUSSION

RESULTS

Plasma lipoprotein concentrations In all patients, VLDL-cholesterol concentrations were 02 mg/100 mL, LDL-cholesterol concentrations were 09 mg/100 mL, and HDL-cholesterol concentrations were 2243 mg/100 mL (x: 29 mg/100 mL). HDL-cholesterol concentrations of the 6 healthy subjects studied ranged from 27 to 45 mg/100 mL (x: 36 mg/100 mL). Plasma carbonyl concentrations The mean carbonyl concentration of the abetalipoproteinemia patients was 0.567 0.031 nmol/mg protein, compared with a reference value of 0.5039 0.0134 nmol/mg protein in healthy, age-matched control subjects (NS). Plasma oxidizability In abetalipoproteinemia patients, the plasma oxidizability lag phase was 28.03 3.16 min and did not differ significantly from that observed in 6 healthy subjects in whom the oxidizability of isolated HDL was measured (24.0 2.79 min). The rate of oxidation also did not differ significantly between the groups: 0.0002 0.000049 and 0.00018 0.000033 OD/min in the patients and healthy subjects, respectively. Plasma low-molecular-weight antioxidants The peak potential of all plasma samples studied was 330 8.3 mV. Because each antioxidant has a characteristic peak potential, this uniformity of plasma peak potential denotes that the same antioxidants were likely present in the plasma of both patients and control subjects. The anodic current of the plasma samples from the patients was 5.227 0.25 A and did not differ significantly from age-matched reference values (5.38 0.20 A).

The neurologic and ophthalmologic symptoms of abetalipoproteinemia are believed to be, at least in part, a consequence of -tocopherol deficiency. -Tocopherol is considered to be the major lipid-soluble antioxidant with an essential role in protecting the integrity of lipid structures, including cell membranes (5, 6). The neurologic manifestations of conditions such as ataxia with vitamin E deficiency, cholestatic liver disease, and abetalipoproteinemia are thus thought to be due to a lack of circulating tocopherol, leading to inadequate protection against oxidative damage (22, 23). In abetalipoproteinemia, not only is the absorption of vitamin E impaired but vitamin E, once absorbed, is associated in plasma with HDL, because the apolipoprotein Bcontaining lipoproteins, which normally are the major carriers of vitamin E in plasma, are almost completely absent (8). Absorption of vitamin A is similarly impaired, and because vitamin A also has antioxidant properties, its deficiency may augment the oxidative stress resulting from vitamin E deficiency. Deficiency of vitamin A in abetalipoproteinemia may also affect retinal changes as a result of the crucial role of vitamin A in phototransduction (9). In the present study, total plasma oxidizability was used as a physiologic model of lipoprotein oxidation. This technique, which measures lipoprotein oxidation in vitro in plasma, accounts not only for lipoprotein oxidation per se but also for the antioxidants in plasma that inhibit lipoprotein oxidation. It is notable that the circulating lipoproteins of patients with abetalipoproteinemia are almost exclusively HDLs. As a result, the oxidation of plasma from patients with abetalipoproteinemia cannot be compared with the oxidation of plasma from subjects with a normal lipoprotein profile (24). We therefore compared the lag phase observed when HDL isolated from the plasma of healthy subjects was oxidized with the lag phase observed when HDL isolated from the plasma of the abetalipoproteinemia patients was oxidized. The lag phase of total plasma oxidation in the abetalipoproteinemia group did not differ significantly from that in the healthy subjects and was, indeed, similar to that previously observed when the oxidation lag phase of isolated HDL was studied (24). Vitamin E in plasma is transported by plasma lipoproteins, and its partitioning between the various lipoprotein fractions is complex. It depends on both total lipid mass ratios and specific vitamin Elipoprotein interactions, as evident by an especially high affinity of vitamin E for both the very small number of apolipoprotein Bcontaining lipoprotein particles present (25) and HDL (26). In abetalipoproteinemia, HDL is the predominant carrier of vitamin E in plasma, which accounts for the extremely low plasma vitamin E concentrations that can be

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achieved even with high-dose vitamin E supplementation. Nevertheless, our results for the HDL oxidation lag phase suggest that this bound vitamin E does provide HDL with adequate protection from oxidation. The susceptibility of HDL to oxidation is particularly sensitive to diet because HDL phospholipids rapidly acquire the fatty acid composition of dietary fat (27). Dietary supplementation with n 6 polyunsaturated fatty acids has been shown to render LDL more susceptible to oxidation, as indicated by a shorter lag phase (28), but to have an opposite effect on the oxidation of HDL. Supplementation with polyunsaturated fatty acids increases HDL2 lag time with no effect on HDL3 lag time, but does not affect the oxidation rate of either LDL or HDL (28). In abetalipoproteinemia patients, plasma and red blood cell membranes are especially deficient in the n 6 polyunsaturated fatty acid linoleic acid (29), as is the HDL of these patients (30). Despite this lower linoleic acid content, however, the HDL lag phase and oxidation rate in our patients did not differ significantly from that in healthy control subjects. Carbonyls result from the modification of amino acids and are an early marker of oxidative damage to proteins (16). Carbonyl concentrations in the plasma of abetalipoproteinemia patients did not differ significantly from concentrations observed in healthy age-matched control subjects. The antioxidant defense system is composed of several endogenous antioxidant enzymes [superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), glutathione peroxidase (EC 1.11.1.9), and glutathione reductase (EC 1.8.1.7)] and lowmolecular-weight antioxidants. Low-molecular-weight antioxidants are both hydrophilic [eg, glutathione, ascorbic acid (vitamin C), NADH, NADPH, uric acid, and polyphenols] and hydrophobic (eg, tocopherols, carotenes, and lycopene). In the face of a decrease in concentration of the major lipophilic antioxidants, vitamin E and vitamin A, and in the presence of chronic oxidative stress, a compensatory increase in hydrophilic antioxidants would be expected. Yet, cyclic voltammetry failed to show any increase in concentration of hydrophilic low-molecular-weight antioxidants in the plasma of patients with abetalipoproteinemia. At the time abetalipoproteinemia is diagnosed, patients are started on vitamin E therapy at a dosage of 100 mg kg 1 d 1 (with dosages in adults of 46 g/d) in combination with vitamin A at a dosage of 23 times the normal requirement (10 00015 000 IU/d; 8). Early treatment appears to abrogate, to a large degree, the neurologic and retinal lesions of abetalipoproteinemia and is believed to attenuate further deterioration of the neurologic and retinal findings already present (7). Nevertheless, we showed previously that progression of retinal changes does occur (9). Although these retinal abnormalities may be the result of factors other than deficiencies of vitamins E and A, including essential fatty acid deficiency, one cannot preclude the possibility that, despite treatment, intratissue antioxidant concentrations are suboptimal and intratissue oxidative damage occurs. Our ability to noninvasively assess antioxidant capacity and antioxidant damage in tissues is obviously limited. On the basis of the plasma carbonyl concentrations, plasma oxidizability, and hydrophilic low-molecular-weight antioxidant capacity measured in the present study, we conclude that enhanced oxidative stress is not apparent in the plasma of abetalipoproteinemia patients receiving long-term supplementation with vitamins E and A.

EG was responsible for initiating the study, analyzing the data, and writing the manuscript. RK was responsible for data analysis and provided the laboratory in which the assays were performed. Neither of the authors had a conflict of interest.

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