Effect of Thyroid Function on LDL Oxidation

From the Centro per lo Studio dell'Ipertensione Arteriosa, delle Dislipidemie e dell'Arteriosclerosi (F. Costantini, S.D.P., D.D.C., F. Cuccurullo, A.M.), and the Thyroid Unit (P.D.R., M.T.G., S.S.), Department of Medicine and Ageing Science, and Institute of Biochemistry (T.B.), University Gabriele D'Annunzio, School of Medicine, Chieti, Italy; the Centro Regionale per L'Arteriosclerosi (G.B.-B., G.C.), Ospedale Civile, Venezia, Italy; and the Clinical Pathology Laboratory of the S.S. Annunziata Hospital, ASL 04 (G.N.), Chieti, Italy.

	Abstract

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Abstract—In this study, the effect of different levels of thyroid hormone and metabolic activity on low density lipoprotein (LDL) oxidation was investigated. Thus, in 16 patients with hyperthyroidism, 16 with hypothyroidism, and 16 age- and sex-matched healthy normolipidemic control subjects, the native LDL content in lipid peroxides, vitamin E, ß-carotene, and lycopene, as well as the susceptibility of these particles to undergo lipid peroxidation, was assessed. Hyperthyroidism was associated with significantly higher lipid peroxidation, as characterized by a higher native LDL content in lipid peroxides, a lower lag phase, and a higher oxidation rate than in the other two groups. This elevated lipid peroxidation was associated with a lower LDL antioxidant concentration. Interestingly, hypothyroid patients showed an intermediate behavior. In fact, in hypothyroidism, LDL oxidation was significantly lower than in hyperthyroidism but higher than in the control group. Hypothyroidism was also characterized by the highest ß-carotene LDL content, whereas vitamin E was significantly lower than in control subjects. In hyperthyroidism but not in the other two groups, LDL oxidation was strongly influenced by free thyroxine blood content. In fact in this group, the native LDL lipid peroxide content and the lag phase were directly and indirectly, respectively, related to free thyroxine blood levels. On the contrary, in hypothyroidism LDL oxidation was strongly and significantly related to serum lipids. In conclusion, both hypothyroidism and hyperthyroidism are characterized by higher levels of LDL oxidation when compared with normolipidemic control subjects. In hyperthyroid patients, the increased lipid peroxidation was strictly related to free thyroxine levels, whereas in hypothyroidism it was strongly influenced by serum lipids.

Key Words: thyroid hormone • lipid peroxidation • LDL oxidation • antioxidant vitamins

	Introduction

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Thyroid hormones play a crucial role in the regulation of mitochondrial oxidative metabolism; the synthesis and degradation of proteins and vitamins, such as vitamin E, vitamin A, and ß-carotene; the sensitivity of tissues to catecholamines; and the regulation of antioxidant enzyme levels.¹ Overt hyperthyroidism and hypothyroidism represent opposite clinical conditions characterized respectively by enhanced oxidative metabolism and reduced lipid and lipoprotein plasma levels and by reduced oxidative metabolism and markedly increased lipid and lipoprotein plasma levels. The hypermetabolic state that characterizes hyperthyroidism should accelerate free radical production in the mitochondria and induce changes in the antioxidant defense system.^{2 3} In contrast, the metabolic suppression brought about by hypothyroidism is associated with a decrease in free radical production, and it has also been suggested that hypothyroidism protects tissues against acceleration of lipid peroxidation.^{3 4 5} Increasing experimental and epidemiological evidence shows that high oxidative stress status favors oxidative modifications of LDL and plays an important role in the development of atherosclerosis.^{6 7 8} Nevertheless, hypothyroidism but not hyperthyroidism represents an important risk factor for atherosclerosis and coronary heart disease.⁹ In view of well-documented strong relationships between blood cholesterol, LDL oxidation, and atherosclerosis, we used two opposite metabolic conditions, overt hyperthyroidism and hypothyroidism, to better understand the relationships between metabolic activity, blood lipids, lipo-protein content, and LDL oxidation.

	Methods

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Sixteen patients with overt hyperthyroidism, 16 with overt hypothyroidism, and 16 control subjects were enrolled in the study. Subjects in accord with inclusion and exclusion criteria were consecutively selected from the population referred to the Regional Thyroid Unit of Chieti, Abruzzo, Italy. Groups were matched for age and sex. All control subjects had normal serum lipid levels, ie, serum TC 5.7 mmol/L and triglycerides 2.8 mmol/L. Overt hyperthyroidism was defined as a basal serum TSH concentration <0.1 µU/mL and a basal serum FT₄ concentration >26 pmol/L. Overt hypothyroidism was defined as a basal serum TSH concentration >20 µU/mL and a basal serum FT₄ concentration <8 pmol/L. The TSH normal range was 0.1 to 4.5 µU/mL, and the FT₄ normal range was 11 to 26 pmol/L. Participants had to fulfill all of the following criteria to be entered into the study: nonsmokers; not taking any medication; not having taken vitamin supplements or other dietary antioxidants for at least 6 months before entry into the study; alcohol intake <1 oz/d; normal plasma glucose levels; normal hepatic and renal function; no evidence of malabsorption; no pancreatic or biliary disease; and no acute medical condition for at least 3 months. All subjects included in the study came from the same geographic area. The mean caloric intake and diet composition were similar among the groups as evaluated by a dietitian who collected diet histories.

Biochemical Determinations
Fasting venous blood samples were obtained for a complete blood count, plasma glucose and protein determinations, and assessment of hepatic and renal function. All of these variables were assayed by standard laboratory techniques. Blood was also obtained for the determination of the lipid profile, serum thyroid hormone concentrations, and LDL isolation. In this study, LDL oxidation was evaluated as the susceptibility of LDL to undergo lipid peroxidation and as the n-LDL lipid peroxide content as assessed by fluorometrically detected TBARS and FPLPs.

Serum Lipids and Lipoprotein Assay
TC and triglycerides were measured by standard automated enzymatic techniques. HDL cholesterol was assessed after precipitation of serum with dextran sulfate. The LDL-C level was calculated by the Friedewald formula: TC-(HDL cholesterol+triglycerides/5).

Serum Hormone Concentrations
The serum TSH concentration was measured by immunoradiometric assay (RIA-gnost hTSH, CIS Bio-international). The serum FT₄ concentration was determined by radioimmunoassay (Coat-A-Count Free T4, Diagnostic Products Corp).

LDL Isolation and Oxidation
After an overnight fast, blood was drawn into test tubes containing 1 mmol/L EDTA. Plasma was separated by low-speed centrifugation at 1500g for 15 minutes, immediately supplemented with 20 µmol/L BHT, stored at -80°C under N₂, and used for LDL isolation within 3 days. Previous studies have shown that plasma storage and freezing/thawing does not affect LDL isolation and its major chemical characteristics.¹⁰ LDL was isolated by single vertical-spin ultracentrifugation¹¹ with a discontinuous NaCl/KBr density gradient.¹² All solutions used for lipoprotein preparations contained 1 mmol/L EDTA. After 86 minutes at 397 700g and 7°C in a Centrikon TVF 6513 vertical rotor (Kontron Instruments), LDL was recovered from the mid-upper part of the gradient and dialyzed in the dark for 22 hours against three changes of PBS, pH 7.4, at 4°C containing 0.01 mmol/L EDTA. LDL protein and LDL-C were determined by established methods.^{13 14} The susceptibility of LDL to undergo lipid peroxidation was assessed spectrophotometrically by continuously monitoring the formation of conjugated dienes at 234 nm.¹⁵ For this purpose, LDL (0.2 mg cholesterol per milliliter) was reconstituted with 5 µmol/L CuSO₄ in PBS, pH 7.4, at 37°C. All determinations were carried out in a computer-assisted diode-array Hewlett-Packard 8452-A spectrophotometer equipped with a seven-position automatic sample changer. The increase in absorbance at 234 nm was recorded every 2 minutes. The lag phase, preceding the formation of conjugated dienes, and the propagation phase, during which time the absorbance at 234 nm rapidly increased to a maximum, were calculated as described previously.¹⁵ The propagation rate was calculated from the slope of the tangent to the absorbance curve during the propagation phase and using a molar extinction coefficient for conjugates dienes (₂₃₄) of 29 500, expressed as nanomoles of dienes formed per minute per milligram of LDL-C.¹⁶

FPLPs in n-LDL
Lipid peroxidation in n-LDL was assessed by measurement of FPLPs.^{17 18 19} FPLPs essentially reflect the interaction of aldehydic lipid peroxidation products with phospholipids and amino groups of the protein.^{17 18 20} The characteristic of these indicators is that they tend to be long-lived and to remain at the sites of oxidative damage.²¹ In brief, the n-LDL sample (1 mL) was diluted with PBS to a final protein concentration of 0.5 mg/mL, mixed with 7 mL chloroform/methanol (2:1, vol/vol) plus water, and briefly centrifuged. The lipid-containing phase was removed, dried under a stream of N₂ gas at room temperature, resuspended in chloroform (2.5 mL), and exposed to UV light. Fluorescence values were estimated spectrofluorometrically at 360 nm excitation and 430 nm emission using a Kontron SFM25 spectrofluor-ometer calibrated with quinine sulfate. Results were expressed as units of relative fluorescence per milligram of LDL-C.

TBARS
The lipid peroxide content of n-LDL was also evaluated fluorometrically as TBARS.²² LDL (100 µg protein) was mixed with 1.5 mL of 0.67% TBA and 1.5 mL of 20% trichloroacetic acid containing 1 mg/mL EDTA. After the mixture was heated at 100°C for 30 minutes, fluorescent reaction products were estimated spectrofluorometrically at 515 nm excitation and 553 nm emission using a Kontron SFM25 spectrofluorometer. Freshly diluted tetramethoxypropane, which yields malondialdehyde, was used as a standard, and results were expressed as nanomoles of malondialdehyde equivalents per milligram of LDL-C.

LDL Antioxidant Determinations
The LDL contents in vitamin E, ß-carotene, and lycopene were determined by HPLC.²³ Vitamins were separated and quantified by using a Kontron system 450 equipped with a UV–visible wavelength variable Kontron detector 430. Analysis was performed by isocratic elution. The flow rate was 1.5 mL/min. The mobile phase, consisting of methanol/butanol/water (89.5:5:5.5, vol/vol/vol), was premixed and vacuum filtered through a 0.45-µm polypropylene membrane filter (Whatman) before use. Autoinjection of 10 µL of organic extract was performed with a Waters autoinjector (model 717 plus autosampler) refrigerated at 5°C. The analytical column used was a replaceable Partisphere 5 C₁₈ cartridge (110x4.7 mm inner diameter, 5-µm particle size; Whatman) protected by a guard cartridge (C₁₈, 5 µm) system and maintained at 45°C. Vitamin E, tocopherol acetate (internal standard), lycopene, and ß-carotene were detected by the UV–visible light spectrophotometer at different wavelengths programmed for analysis as follows: at 0 minutes, 290 nm; 4.5 minutes, 280 nm; and 15 to 22 minutes, 450 nm. Vitamins were expressed as micrograms per milligram of LDL-C.

Free Fatty Acid Measurements in LDL
The fatty acid composition of LDL was determined by HPLC according to the method of Miwa and Yamamoto.²⁴ In brief, 1 mL LDL was extracted with 8 mL chloroform/methanol (2:1, vol/vol). The extracts were washed with 2 mL of HPLC water and dried under N₂; 25 µL of ethanol containing 2 nmol/L margaric (heptadecanoic) acid as the internal standard was then added. Fatty acids were converted into their 2-nitrophenylhydrazide forms by the addition of 100 µL of 2-nitrophenylhydrazine HCl (20 mmol/L) and 200 µL of equal volumes of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (250 mmol/L) and 13% ethanolic pyridine. The mixture was then heated at 60°C for 20 minutes. After the addition of 50 µL of KOH solution (KOH 15% in methanol/water, 4:1, vol/vol), the mixture was heated further at 60°C for 15 minutes. To the resulting mixture of hydrazides, 2 mL of 1/30 mol/L phosphate buffer (pH 6.4)–0.5 mol/L HCl (3.8:0.4, vol/vol) and 1.5 mL of n-hexane were added. After vortexing and centrifugation, the n-hexane layer was taken and evaporated under N₂. The residue was dissolved in 50 µL methanol, and aliquots were injected into the HPLC utilizing a Supelcosil LC-8 reversed-phase column. The samples were eluted with acetonitrile/water (85:15, vol/vol, pH 4.5), and the chromatograms were recorded with a Perkin-Elmer Sigma 15 data station utilizing a Perkin-Elmer LC-75 monitor set at 400 nm.

Statistical Analysis
Data are reported as mean±SD. Differences between the three groups were analyzed by one-way ANOVA, followed by the Scheffé test for multiple comparisons between groups. Simple linear regression analysis was also used where appropriate. Statistical significance was defined as P<.05. Statistical analyses were performed using STATVIEWS software (Abacus Concepts Inc) for the Apple Macintosh computer.

	Results

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Clinical and Laboratory Data
Serum FT₄ and TSH concentrations are reported in Table 1. Serum TC, LDL-C, and triglycerides were significantly higher in hypothyroid patients when compared with either hyperthyroid patients or control subjects (Table 1). Hyperthyroid patients showed lower TC, LDL-C, and triglyceride serum contents than the control group (Table 1). HDL cholesterol was significantly higher in hypothyroid and control groups when compared with the hyperthyroid group; no significant difference was present between hypothyroid patients and control subjects.

Lipid Peroxidation Indices
Hyperthyroidism was associated with significantly higher FPLP and TBARS n-LDL contents than in the other two groups (Table 2). Hypothyroid patients showed significantly higher FPLP and TBARS n-LDL content than control subjects (Table 2). LDL susceptibility to oxidation was significantly increased in hyperthyroid patients when compared with the other two groups (Table 2). In hypothyroid patients, the lag phase was shorter than in the control group, but this difference was not statistically significant. Interestingly, when the oxidation rate was taken into account, both hyperthyroid and hypothyroid groups showed significantly higher values than the control group, but no significant difference was found between hyperthyroidism and hypothyroidism. The cholesterol to protein ratio (Table 2) was significantly higher in the hypothyroid group than in the other two groups. This ratio was not significantly different between hyperthyroid and control groups.

LDL Content in Antioxidant Vitamins
Vitamin E LDL content was significantly lower in the hyperthyroid group than in the other two groups (Table 3). The hypothyroid group showed significantly lower vitamin E LDL contents than did control subjects. The ß-carotene LDL content was significantly higher in the hypothyroid group than in the other two groups (Table 3); moreover, ß-carotene was significantly lower in the hyperthyroid group than in the control group. Lycopene LDL level was significantly lower in hyperthyroid patients than in control subjects; no significant difference was found between hypothyroid and control groups (Table 3) or between hypothyroid and hyperthyroid groups.

LDL Composition in Free Fatty Acids
Hyperthyroidism showed a significantly higher relative arachidonic acid LDL content than the other two groups (Table 4). Hypothyroidism was characterized by a significantly higher LDL total fatty acid content than the other two groups (Table 4). The control group showed a significantly higher relative oleic acid content than the other two groups (Table 4). Linoleic acid LDL content was significantly higher in hypothyroidism than in the control group, but no significant difference was found between hyperthyroidism and hypothyroidism as well as between hyperthyroidism and control (Table 4). Eicosapentaenoic acid was significantly higher in the hypothyroid group than in control subjects; no significant difference was observed between hyperthyroid and hypothyroid groups or between the hyperthyroid group and control subjects. Docosahexaenoic acid was significantly higher in hyperthyroid patients than in the other two groups but similar between the hypothyroid and control groups. The ratio of the LDL content in oleic to that in linoleic acid was significantly lower in hyperthyroid and hypothyroid groups than in the control group; no significant difference was found between hyperthyroidism and hypothyroidism (Table 4).

Correlations
In hyperthyroid patients, the n-LDL content in FPLPs and TBARS and the oxidation rate values were significantly and directly related to FT₄ serum levels (r=.87, P=.0001, Fig 1; r=.80, P<.001; and r=.56, P<.05). Moreover, in this group, FT₄ concentration was also significantly and inversely related to the lag phase (r=-.93, P=.0001, Fig 1). On the contrary, no significant correlation was present between FT₄ and LDL oxidation indices in the hypothyroid group.

View larger version (17K): [in this window] [in a new window]   Figure 1. In hyperthyroid patients the lag phase and LDL content in lipid peroxides (FPLPs) are significantly related to serum FT4 levels.

In the control group a weak but nonsignificant correlation was present between FT₄ values and n-LDL contents in FPLPs, TBARS, the lag phase, and the oxidation rate. No significant relationship was found between FT₄ and LDL contents in vitamin E, ß-carotene, and lycopene in the three groups. In the hypothyroid group, TC and LDL-C were significantly and directly related to n-LDL content in FPLPs (r=.75, P<.01, Fig 2; r=.74, P<.01) and TBARS (r=.74, P<.01; r=.73, P<.01). In this group, TC and LDL-C were also significantly and inversely related to the lag phase (r=-.53, P<.05; r=-.59, P<.05, Fig 2). On the contrary, in hyperthyroid and control groups, TC, and LDL-C were not significantly related to FPLPs, TBARS, and the lag phase.

View larger version (15K): [in this window] [in a new window]   Figure 2. In the hypothyroid group the lag phase and LDL content in lipid peroxides (FPLPs) are significantly related to LDL-C plasma levels.

In all groups the n-LDL content in FPLPs and TBARS was significantly and inversely related to the lag phase (hyperthyroidism, r=-.86, P<.01 and r=-.82, P<.01; hypothyroidism: r=-.68, P<.01 and r=-.72, P<.01; and control subjects: r=-.60, P<.05 and r=-.58, P<.05). Vitamin E LDL content was significantly and directly related to the lag phase in all groups (hyperthyroid group: r=.54, P<.05; hypothyroid group: r=.54, P<.05; and control group: r=.58, P<.05). Vitamin E LDL content was also significantly and inversely related to n-LDL content in FPLPs and TBARS in all groups (hyperthyroidism: r=-.52, P<.05 and r-0.53, P<.05; hypothyroidism: r=-.52, P<.05 and r=-.54, P<.05; and control subjects: r=-.50, P=.05 and r=-.54, P<.05). Finally, the oxidation rate was significantly and inversely related to the oleic to linoleic acid ratio in hyperthyroid and hypothyroid groups (r=-.66, P<.01; r=-.68, P<.01).

	Discussion

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Our study confirms that LDL oxidation is markedly higher in hyperthyroidism than in hypothyroidism or control subjects. The increased oxidative stress was associated with higher consumption of lipid-soluble antioxidant vitamins. Accordingly, it is well known that hyperthyroidism accelerates mitochondrial oxidative metabolism, resulting in increased free radical production and lipid peroxidation.^{2 3} Moreover, inspection of the fatty acid composition of LDL particles reveals a higher relative arachidonic acid content in this group (Table 4). Arachidonic acid is a polyunsaturated fatty acid with four double bounds, which are easily oxidized and thus contribute to increase lipid peroxidation.²⁵

Unexpectedly, hypothyroid patients showed higher lipid peroxidation than did healthy normocholesterolemic control subjects. This higher oxidative stress was characterized by a significantly higher LDL content in preformed lipid peroxides and a higher oxidation rate, whereas the lag phase was shorter but not statistically different than in control subjects. Interestingly, hypothyroid patients showed oxidation rates not significantly different from those found in the hyperthyroid group, despite significantly longer lag phases. These data suggest that in hypothyroidism the LDL oxidation process is delayed compared with that in the hyperthyroid group, but once the autocatalytic chain reaction of lipid peroxidation begins, it propagates very quickly inside the particle. This phenomenon is difficult to explain. In hypothyroid patients, vitamin E LDL content was significantly lower than in control subjects, whereas the ß-carotene LDL level was significantly higher. Reportedly, the higher-than-normal ß-carotene LDL content found in hypothyroidism can be explained by a blockage of ß-carotene conversion to vitamin A due to the lack of thyroid hormone.²⁶ It has recently been reported that ß-carotene has the ability to "quench" singlet oxygen and can also act as a lipophilic, chain-breaking antioxidant.²⁷ However, recent studies have shown that in vivo supplementation with large doses of ß-carotene over the long term does not result in an increased LDL resistance to Cu²⁺-induced oxidation.^{28 29 30 31} Accordingly, in our study vitamin E but not ß-carotene LDL content was significantly and directly related to the lag phase and indirectly related to FPLPs and TBARS in all groups. The quite normal lag phase found in the hypothyroid group, despite the lower vitamin E and higher preformed lipid peroxide LDL contents, might indicate that ß-carotene was able to increase LDL resistance to oxidation, especially because it is primarily localized in the core of LDL,³² along with the highly vulnerable polyunsaturated fatty acid–rich cholesterol esters. Nevertheless, it has recently been hypothesized that ß-carotene can auto-oxidize via a peroxyl radical mechanism.³³ These peroxyl radicals can then initiate attack on other ß-carotene molecules, giving rise to a chain reaction comparable to that observed during lipid peroxidation. Spontaneous auto-oxidation is suppressed by -tocopherol. Since during the lag phase LDL becomes progressively depleted of its antioxidants, with -tocopherol as the first one and ß-carotene as the last,³⁴ we speculate that when vitamin E is completely exhausted, a pro-oxidant rather than an antioxidant effect of ß-carotene could prevail. Thus, lack of effective antioxidant protection as well as a possible pro-oxidant activity of elevated ß-carotene LDL content³⁵ could explain the higher-than-normal LDL oxidation observed in hypothyroid patients. Moreover, the higher oxidation rate found in hypothyroid and hyperthyroid than in control subjects might also be explained by the lower oleic to linoleic acid ratio present in the former group. In fact, it has recently been demonstrated that the oleic to linoleic acid ratio is inversely correlated with the oxidation rate.²⁵

Interestingly, LDL oxidation seemed to be affected by thyroid function. However, this influence seems to become crucial only in the presence of very high levels of thyroid hormone (Fig 1). In contrast, in hypothyroidism but not in hyperthyroidism or normocholesterolemia, LDL oxidation was strongly influenced by TC and LDL-C serum contents. In fact, in these patients the markedly elevated blood concentrations of TC and LDL-C were significantly and directly related to LDL susceptibility to oxidation (Fig 2). Accordingly, it has been demonstrated that hyperlipidemic subjects show significantly higher LDL oxidation than do normolipidemic control subjects.^{36 37} Hypercholesterolemic subjects have not only higher concentrations of LDL but also substantially "older" LDL due to decreased cellular receptor numbers and consequent reduced removal of LDL from plasma.³⁸ Aging lipoproteins are subjected to prolonged residence time and are repeatedly exposed to a variety of oxidizing species that could allow formation and accumulation of lipid peroxidation products, thus enhancing their susceptibility to oxidation.^{37 38} It is well known that hypothyroidism leads to decreased activity of the LDL receptor, which contributes to hypercholesterolemia.³⁹ Thus, in hypothyroidism a higher cholesterol to protein ratio⁴⁰ together with an increased age of LDL could contribute to the enhanced oxidizability of these particles.

In conclusion, a marked increase of metabolic activity can favor a pro-oxidant environment capable of significantly increased antioxidant consumption and LDL oxidation. Nevertheless, high serum cholesterol levels seem to represent a very strong pro-oxidant factor that can also favor LDL oxidative modification in patients with a very low metabolic rate, such as in hypothyroidism. Finally, considering the different prevalence of atherosclerosis in hyperthyroidism and hypothyroidism,⁹ we can speculate that without a sufficiently high blood cholesterol content, even a very high level of oxidative stress is rarely associated with atherosclerotic lesions.

	Selected Abbreviations and Acronyms

 

FPLP = fluorescent product of lipid peroxidation FT4 = free thyroxine HPLC = high-performance liquid chromatography LDL-C = LDL cholesterol n- = native TBARS = thiobarbituric acid–reactive substances TC = total cholesterol TSH = thyrotropin

	Acknowledgments

 
The skillful technical assistance given by Guerino Di Sciascio, Corrado Romano, and Maria Di Riti is gratefully acknowledged. We thank Dr Rossella Tonelli for her help in the preparation of the manuscript.

	Footnotes

 
Reprint requests to Prof Andrea Mezzetti, MD, Dipartimento di Medicina e Scienze dell'Invecchiamento, Policlinico S.S. Annunziata, Via dei Vestini, 66013-Chieti Scalo, Italy.

Received May 13, 1997; accepted December 3, 1997.

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