| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Lipids, Lipoproteins, and Cardiovascular Risk Factors |
1 Department of Core Clinical Pathology and Biochemistry, PathWest Laboratory Medicine WA, Royal Perth Hospital, Perth, WA, Australia.
2 School of Medicine and Pharmacology, and 3
School of Surgery and Pathology, University of Western Australia. Crawley, WA, Australia.
aAddress correspondence to this author at: Department of Core Clinical Pathology and Biochemistry, PathWest Laboratory Medicine WA, Royal Perth Hospital, GPO Box X2213, Perth, WA 6847, Australia. Fax 61 (08) 9224-1789; e-mail john.burnett{at}health.wa.gov.au.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-tocopherol and decreased LDL-cholesterol and apolipoprotein (apo) B. We examined the effect of truncated apoB variants on vitamin E metabolism and oxidative stress in persons with FHBL.
Methods: We studied 9 individuals with heterozygous FHBL [mean (SE) age, 40 (5) years; body mass index (BMI), 27 (10) kg/m2] and 7 normolipidemic controls [age, 41 (5) years; BMI, 25 (2) kg/m2]. We also studied 3 children—2 with homozygous FHBL (apoB-30.9) and 1 with abetalipoproteinemia—who were receiving -tocopherol supplementation. We used HPLC with electrochemical detection to measure - and 
-tocopherol in serum, erythrocytes, and platelets, and gas chromatography–mass spectrometry to measure F2-isoprostanes and tocopherol metabolites in urine as markers of oxidative stress and tocopherol intake, respectively.
Results: Compared with controls, persons with FHBL had significantly lower fasting plasma concentrations of total cholesterol [2.4 (0.2) vs 4.7 (0.2) mmol/L], triglycerides [0.5 (0.1) vs 0.9 (0.1) mmol/L], LDL-cholesterol [0.7 (0.1) vs 2.8 (0.3) mmol/L], apoB [0.23 (0.02) vs 0.84 (0.08) g/L], -tocopherol [13.6 (1.0) vs 28.7 (1.4) µmol/L], and -tocopherol [1.0 (0.1) vs 1.8 (0.3) µmol/L] (all P <0.03). Erythrocyte -tocopherol was decreased [5.0 (0.2) vs 6.0 (0.3) µmol/L; P <0.005], but we observed no differences in lipid-adjusted serum tocopherols, erythrocyte -tocopherol, platelet - or -tocopherol, urinary F2-isoprostanes, or tocopherol metabolites.
Conclusion: Taken together, our findings do not support the recommendation that persons with heterozygous FHBL receive vitamin E supplementation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Persons heterozygous for FHBL are usually asymptomatic but have plasma LDL-cholesterol and apoB concentrations that are only one fourth to one third of reference values. Other clinical manifestations are serum -tocopherol concentrations below or at the lower limit of the reference interval and abnormal erythrocyte morphology with characteristic acanthocytes, as seen in persons with vitamin E deficiency (4). The clinical and biochemical features in homozygous and compound heterozygous FHBL can include acanthocytosis, deficiencies of fat-soluble vitamins secondary to malabsorption, an atypical form of retinitis pigmentosa, and neuromuscular abnormalities. Retinitis pigmentosa and neuromuscular abnormalities are related primarily to deficiencies in fat-soluble vitamins, especially vitamins A and E, resulting from their impaired absorption and transport.
Abetalipoproteinemia (ABL; OMIM 200100) is a very rare autosomal recessive disorder of lipoprotein metabolism in which sequence variations in the microsomal triglyceride transfer protein (MTP) gene lead to virtually undetectable plasma concentrations of apoB-containing lipoproteins. Microsomal triglyceride transfer protein is a molecular chaperone that facilitates the assembly and secretion of triglyceride-rich apoB-containing lipoproteins, namely, chylomicrons and VLDL. Patients often present in childhood or early infancy with failure to thrive, fat malabsorption, and low plasma cholesterol and vitamin E concentrations.
Vitamin E is essential for neurologic function and is transported in plasma in association with the apoB-containing lipoproteins. Acanthocytes typically comprise 50% to 100% of erythrocytes in ABL and may result from vitamin E deficiency or altered membrane lipid composition. Long-term high-dose vitamin A and E supplementation should prevent or slow progression of the neuromuscular and retinal degenerative disease (5). Persons with compound heterozygous FHBL are often clinically indistinguishable from those with ABL(1)(2)(3).
In persons with FHBL, tocopherol delivery to tissues occurs primarily through chylomicron and HDL metabolism (6). Vitamin E supplementation is thought to be safe, and its use has been recommended for management of FHBL(4). Results of a recent metaanalysis, however, suggest that supplementation with 
400 IU of -tocopherol per day might increase the risk of heart failure(7) or all-cause mortality(8) in certain population groups. It is not known whether this risk affects persons with low plasma cholesterol, nor is it known whether tocopherol isomer transport is sufficient to prevent vitamin E deficiency or complications associated with oxidative stress in persons with heterozygous FHBL.
We used the relationship between plasma and cellular tocopherol concentrations and urinary markers of tocopherol intake and oxidative stress as a means to investigate the effects of truncated apoB variants in FHBL on vitamin E metabolism.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-tocopherol supplementation. The heterozygous FHBL adults were identified after referral to a lipid disorders clinic for investigation of hypocholesterolemia. The homozygous FHBL and ABL children were identified after referral to a pediatric gastroenterology clinic for investigation of failure to thrive. Baseline characteristics of the study participants are provided in Tables 1
and 2
.
|
|
Blood and spot morning urine samples were collected from each participant after a 10-h overnight fast. Venous blood was collected into 3-mL serum and 9-mL EDTA tubes and processed immediately. The serum tube was centrifuged at 2000g for 10 min at 4 °C, and the serum was then frozen at –80 °C until analysis. The EDTA tubes were used for platelet and erythrocyte analyses. Urine samples were placed in 5-mL tubes without a preservative and stored at –80 °C until analysis.
Approval for this study was obtained from the Royal Perth Hospital Ethics Committee, and informed consent was received from the participants, or the parents in the case of the 3 children.
platelet preparation
Platelets were prepared for tocopherol analysis as described previously (9) with minor modifications. In brief, the samples were centrifuged at 200g for 15 min at 4 °C, the platelet-rich plasma fraction was removed, and platelets were counted with an Abbott CELL-DYN 4000 Hematology analyzer. After cell counting, 500-µL aliquots of the platelet-rich plasma fraction were dispensed into Eppendorf microcentrifuge tubes and centrifuged at 2000g for 10 min at 4 °C. Platelets were washed 3 times in a citrate saline solution (1.7 g/L trisodium citrate dihydrate, 8.7 g/L NaCl), after which 100 µL of HPLC-grade ethanol was added to the remaining pellet and the sample was stored at –80 °C until analysis.
erythrocyte preparation
Erythrocytes were prepared for tocopherol analysis as described previously (9). In brief, erythrocytes were washed 3 times in saline (9 g/L NaCl) solution containing 10 g/L pyrogallol and resuspended in this solution to give a hematocrit of 
50%. The exact hematocrit was then determined, and 500-µL aliquots of washed erythrocytes were stored at –80 °C until analysis.
hplc analysis of tocopherols
Serum tocopherols were extracted as described previously (10) with minor modifications. In brief, 200 µL of serum in a borosilicate glass tube was enriched with 50 µL of tocol (10 mg/L) and mixed for 20 s. We then added 200 µL of ethanol, mixed the serum again for 60 s, and left it in the dark for 5 min. The sample was extracted with 1 mL of hexane, vortex-mixed for 3 min, and then centrifuged at 2700g for 10 min at 4 °C. A 600-µL portion of the hexane was removed and evaporated under nitrogen, and the residue was reconstituted in 200 µL of methanol. We injected 1 µL of this reconstituted sample on the column.
Platelet tocopherols were extracted as described previously (11) with minor modifications. In brief, the prepared platelet pellets were sonicated with a Branson Sonifier 150 for 5 quick pulses with the power setting at 3. We then added 800 µL of hexane to the pellets, followed by 50 µL of tocol (1 mg/L). The samples were mixed for 60 s and then centrifuged at 2700g for 10 min at 4 °C. A 600-µL portion of hexane was removed and evaporated under nitrogen, and the residue was reconstituted in 150 µL of methanol. We injected 15 µL of the reconstituted sample on the column.
Erythrocyte tocopherols were extracted as described previously (9) with minor modifications. In brief, we added 500 µL of ethanol to 500 µL of washed erythrocytes, mixed them for 20 s, enriched the samples with 50 µL of tocol (10 mg/L), and mixed the sample again for 20 s. We then added 2 mL of hexane and mixed for 60 s, before centrifugation at 2700g for 10 min at 4 °C. We removed a 1-mL portion of the hexane phase and dried it under nitrogen. We reconstituted the residue in 300 µL of methanol and injected 15 µL of this reconstituted sample on the column.
Tocopherols were separated by HPLC on an Agilent 1100 HPLC equipped with a Lichrospher 100 RP-18 column [125 x 4.6 mm (i.d.); bead size, 5 µm]. The mobile phase was 990 mL/L methanol and 10 mL/L water containing 10 µmol/L lithium acetate, and the flow rate was 1 mL/min. Tocol was used as an internal standard. Detection was performed on an ESA Coulochem IIITM coulometric electrochemical detector set at 600 V and 1 µA for each assay. All reagents were HPLC grade, and the calibrators were obtained from Calbiochem.
In-house quality-control samples were assayed in duplicate at the beginning, middle, and end of each assay. The inter- and intraassay CVs for the tocopherol methods were <10%. All measurements were performed in duplicate, and samples with a duplicate CV >10% were reassayed. The extraction recovery for - and -tocopherols was 95%–105% for serum, erythrocytes, and platelets. The functional sensitivities for - and -tocopherol in serum, packed erythrocytes, and platelets were, respectively, 1.0 µmol/L, 0.3 µmol/L, and 0.03 nmol/109 cells. These values, obtained by assaying multiple samples across the linear range of the assay, were defined as the lowest reportable concentration of the analyte giving a CV <10% for the samples assayed in triplicate. Repeat analyses showed that - and -tocopherols were stable for at least 6 months at –80 °C, and all samples were assayed within 4 months of collection.
gas chromatographic–mass spectrometric analysis of tocopherol metabolites
We measured the urinary tocopherol metabolites with gas chromatography–mass spectrometry (GC-MS) (12) with minor modifications. In brief, we added 50 µL of the internal standard, deuterium-labeled 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman (d9--CEHC; 0.1 mmol/L in ethanol) to 1 mL of freshly thawed urine and then added 1 mL of 0.33 mol/L potassium phosphate buffer (pH 7.4) containing 350 U of Escherichia coli ß-glucuronidase (type IX-A). The urine was incubated for 3.5 h at 37 °C in the dark, and then acidified with 0.25 mL of 6 mmol/L HCl and extracted with 8 mL of hexane–tert-butyl methyl ether (4:1 by volume). Urine extracts were dried under nitrogen, sialated by reaction with 50 µL of anhydrous pyridine and 50 µL of N,O-bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + TMCS), and then heated at 60 °C for 1 h. Samples were injected directly into an Agilent 5973 GC-MS equipped with a DB-5MS column [25 m x 0.2 mm (i.d.); 0.33 µm film thickness]. The initial oven temperature of 160 °C was held for 0.5 min and then increased at 20 °C/min to 300 °C, with a final hold at 300 °C for 10 min. The carrier gas was helium. Selected-ion monitoring of the molecular ion and one major fragment ion (as a qualifying ion) of each metabolite was as follows: internal standard, d9--CEHC, m/z 431 and 246; -CEHC, m/z 422 and 237; -CEHC, m/z 408 and 223; and 
-CEHC, m/z 394 and 209(13). Quality-control samples were run with each assay, and the inter- and intraassay CVs were <10%.
f2-isoprostanes
Free F2-isoprostanes were measured in spot urine samples by GC-MS (14). Quality-control samples were run with each assay, and the inter- and intraassay CVs were <10%.
lipid analysis
Total cholesterol, triglycerides, and HDL-cholesterol were measured with enzymatic, colorimetric assays [cholesterol oxidase/p-aminophenazone (CHOD-PAP; HiCo), glycerol-3-phosphate oxidase/p-aminophenazone (GPO-PAP), and direct method HDL-Plus, respectively] from Roche Diagnostics on a Hitachi 917 chemistry analyzer. LDL-cholesterol was calculated according to the Friedewald equation (15). ApoA-I and apoB concentrations were measured on a Behring BN-II nephelometer. For lipid and apolipoprotein assays, inter- and intraassay CVs were <6% and <10%, respectively.
statistical analysis
All data are presented as the mean (SE). Mean comparisons were performed with SPSS for Windows, Ver. 12 (SPSS Inc.). A P value <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Plasma HDL-cholesterol and apoA-I concentrations were not different between the 2 groups. Serum -tocopherol (–53%), and -tocopherol (–44%) concentrations were significantly lower in heterozygous FHBL individuals than in controls (all P <0.03; Table 3
). Although erythrocyte -tocopherol concentrations were decreased (–17%) in heterozygous FHBL individuals, we observed no differences between groups for lipid-adjusted serum tocopherols, erythrocyte -tocopherol, platelet - or -tocopherol, or urinary F2-isoprostanes and tocopherol metabolites.
|
The characteristics and analytical results from the 3 children (2 with homozygous FHBL and 1 with ABL) receiving -tocopherol supplementation are shown in Table 2
. Plasma lipid profiles were similar in the 2 homozygous FHBL patients, but homozygous FHBL patient 1 was receiving a 2-fold higher daily dose of -tocopherol, which was reflected in the increased platelet -tocopherol and urinary -CEHC concentrations. This finding might in part explain the low urinary F2-isoprostane concentrations in this patient. Homozygous FHBL patient 2 received the lowest dose of -tocopherol supplement, and this was reflected in lower platelet -tocopherol and urinary -CEHC concentrations. This patient had the highest F2-isoprostanes of all study participants and also had very low concentrations of urinary -CEHC, possibly reflecting a low dietary intake of -tocopherol.
The ABL patient had a lipid profile similar to those of the 2 homozygous FHBL patients but, as predicted, had low serum and cellular tocopherol concentrations. The increased urine metabolites might reflect high-dose -tocopherol supplementation in this individual. Moreover, the urinary output of F2-isoprostanes was greater than in the controls but less than in the homozygous FHBL children receiving the lower dose supplement.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- and -tocopherol. Although erythrocyte -tocopherol concentrations were decreased in heterozygous FHBL persons, no differences were observed for lipid-adjusted serum tocopherols, erythrocyte -tocopherol, platelet - or -tocopherol, or urinary F2-isoprostanes and tocopherol metabolites. Taken together, our findings do not support the hypothesis that heterozygous FHBL persons require vitamin E supplementation. The search for an appropriate marker of tocopherol status in persons with low cholesterol has been difficult. Tocopherol is commonly measured in plasma, and plasma and adipose tissue tocopherols have been reported to be equally good markers of intake (16). The challenge arises when plasma lipid concentrations are low, lipoprotein transport pathways are saturated, and serum tocopherol concentrations are decreased.
One approach has been to measure cellular tocopherol concentrations to assess tocopherol transport in vivo. Because erythrocytes are in the circulation for 120 days, they might reflect long-term intake. However, erythrocyte tocopherol concentrations have been shown to passively reflect lipid concentrations (11), a finding that could explain the lower erythrocyte -tocopherol concentrations in heterozygous FHBL persons. In vitro erythrocyte-to-HDL exchange of -tocopherol has been described(17) and might contribute to the observed reduction in erythrocyte -tocopherol concentrations. Although platelet tocopherol concentrations have not been demonstrated to correlate with those in peripheral nervous tissue, platelet concentrations might better reflect dietary vitamin E intake because they are not passively influenced by lipid concentrations(18). Our preference, therefore, was to use platelet tocopherol concentrations to assess in vivo tocopherol transport. Platelet tocopherols were not different between the heterozygous FHBL patients and the controls.
Tocopherol metabolites in urine are markers of short-term tocopherol intake (12)(19). Measurement of urinary F2-isoprostanes, a marker of in vivo lipid peroxidation(20)(21), also provides an assessment of daily isoprostane excretion in humans(22). Tocopherol metabolites were not different between the heterozygous FHBL persons and controls, consistent with similar dietary intake of tocopherols in the 2 groups. The lack of a difference in urinary F2-isoprostane excretion between the heterozygous FHBL persons and the controls indicates a lack of oxidative stress in heterozygous FHBL individuals.
Recent studies have demonstrated the potential importance of -tocopherol in the regulation of specific genes (23); it therefore follows that a decrease in cellular -tocopherol may have clinical consequences. Although erythrocyte -tocopherol was decreased, we observed no differences between groups for lipid-adjusted serum tocopherols, erythrocyte -tocopherol, platelet - or -tocopherol, or urinary F2-isoprostanes and tocopherol metabolites. These findings suggest that adequate delivery of tocopherols to cells and the inhibition of oxidative stress in vivo occur via the chylomicron remnant and HDL metabolic pathways.
Our examination of tocopherol status and oxidative stress in 2 children with homozygous FHBL and 1 child with ABL, all receiving vitamin E supplementation, revealed moderately increased erythrocyte -tocopherol concentrations compared with controls in the homozygous FHBL children. Platelet -tocopherol concentrations did not differ in the 2 homozygous FHBL children, but there were 2- to 3-fold increases in these children compared with the controls. This finding, particularly in the homozygous FHBL child receiving the higher dose of vitamin E, is consistent with the notion that platelet tocopherol concentrations provide the best measure of cellular tocopherol transport and reflect intake. The measurement of urinary metabolites can also provide important information regarding compliance to therapy, as seen in these children, because the highest dose of tocopherol leads to the highest output for urine -CEHC. Although their lipid phenotypes may be similar, patients with homozygous FHBL and ABL are not identical and might differ in lipoprotein kinetics. Our data highlight the importance of using more than one index of tocopherol intake to evaluate treatment efficacy in patients with marked hypocholesterolemia.
Recent studies have also examined the efficacy of vitamin supplementation in persons with ABL or homozygous FHBL (5)(24). Retinal changes were evident in persons with ABL and homozygous FHBL, despite early treatment with high-dose oral vitamin A and E(24). Oxidative stress was not assessed in this study, however. In another study, oxidative stress was measured in persons with ABL who had received high-dose vitamin E and A supplementation since infancy, and measurement of plasma carbonyls and the lag phase for the oxidization of plasma and HDL did not provide evidence for oxidative stress(5).
In one of the homozygous FHBL patients in our study, urinary F2-isoprostanes were decreased, whereas in the other they were markedly increased. This finding might be attributable to the age difference between the 2 children, the dose of -tocopherol, or the contribution from dietary sources of vitamin E. It has been shown that babies have high concentrations of urinary isoprostanes (25), possibly because of oxidative stress at birth and the low efficiency of natural antioxidant systems in the newborn(26). Of note, in our study the homozygous FHBL child with increased urinary F2-isoprostanes was only 6 months old and had very low concentrations of the -CEHC metabolite of -tocopherol, indicating decreased dietary intake of this nutrient.
There has been considerable interest in the potential benefits of -tocopherol for human health (27)(28), and a recent analysis suggested that various forms of vitamin E may be important in the preservation of cognition in persons with Alzheimer disease(29). Clearly, further investigation is needed to assess the efficacy of the different forms of vitamin E and their relative effects on oxidative stress in persons with very low cholesterol. Amid recent interest in the potential benefits of tocotrienols and neurologic function(29), there remains potential for different forms of vitamin E to further reduce sequelae in conditions associated with low cholesterol and oxidative stress.
In summary, we examined in vivo serum and cellular tocopherol concentrations and oxidative stress in persons with heterozygous FHBL. Taken together, in the absence of clinical signs associated with vitamin E deficiency, our findings do not support the theory that heterozygous FHBL individuals require vitamin E supplementation.
|
Acknowledgments |
|---|
-CEHC. This work was supported by grants from the Royal Perth Hospital Medical Research Foundation, the Raine Medical Research Foundation, the National Health & Medical Research Council (403908), and the National Heart Foundation of Australia (G 139 1155). |
Footnotes |
|---|
2 Human genes: APOB, apolipoprotein B; and MTP, microsomal triglyceride transfer protein.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am J Clin Nutr 1979;32:2143-2149.- and -tocopherol in human plasma. Free Radic Biol Med 2002;32:333-340.[CrossRef][Medline]
[Order article via Infotrieve]- and -tocopherol in plasma and adipose tissue as biomarkers of intake in Costa Rican adults. Am J Clin Nutr 2001;74:356-363.-tocopherol-enriched HDL and erythrocytes: application to asymptomatic hypercholesterolemic men. Free Radic Biol Med 2000;28:815-823.[CrossRef][Medline]
[Order article via Infotrieve]-tocopherol in humans. J Lipid Res 1999;40:665-671.-tocopherol. Biol Chem 2004;385:585-591.[Medline]
[Order article via Infotrieve]-Tocopherol, the major form of vitamin E in the U S diet, deserves more attention. Am J Clin Nutr 2001;74:714-722.-Tocopherol traps mutagenic electrophiles such as NO(X) and complements -tocopherol: physiological implications. Proc Natl Acad Sci U S A 1997;94:3217-3222.The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  J. R. Burnett, S. Zhong, Z. G. Jiang, A. J. Hooper, E. A. Fisher, R. S. McLeod, Y. Zhao, P. H. R. Barrett, R. A. Hegele, F. M. van Bockxmeer, et al. Missense Mutations in APOB within the beta{alpha}1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia J. Biol. Chem., August 17, 2007; 282(33): 24270 - 24283. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
