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Baseline Diene Conjugation in LDL Lipids as a Direct Measure of In Vivo LDL Oxidation
Clinical Biochemistry, Vol. 31, No. 4, 257–261, 1998 Copyright © 1998 The Canadian Society of Clinical Chemists Printed in the USA. All rights reserved 0009-9120/98 $19.00 1 .00
PII S0009-9120(98)00018-6
Baseline Diene Conjugation in LDL Lipids as a Direct Measure of In Vivo LDL Oxidation MARKKU AHOTUPA,1 JUKKA MARNIEMI,2 TERHO LEHTIMA¨KI,3 KATI TALVINEN,1 OLLI T. RAITAKARI,4 TOMMI VASANKARI,1 JORMA VIIKARI,5 JUKKA LUOMA,6 and SEPPO YLA¨-HERTTUALA6 1
MCA Research Laboratory, Department of Physiology, University of Turku, Turku, Finland, Research and Development Centre, Social Insurance Institution, Turku, Finland, 3Department of Clinical Chemistry, University Hospital of Tampere and Department of Medical Biochemistry, Medical School, University of Tampere, Tampere, Finland, 4Department of Clinical Physiology, University Hospital of Turku, Turku, Finland, 5Department of Internal Medicine, University Hospital of Turku, Turku, Finland, and 6A. I. Virtanen Institute, Kuopio, Finland 2
Objectives: To additionally test validity of the recently developed method (LDL baseline diene conjugation, LDL-BDC) for determination of circulating oxidized LDL. Design and methods: A detailed comparison between the ultracentrifugation and heparin precipitation methods for LDL isolation was performed to test suitability of the fast precipitation method. Validity of LDL-BDC as an indicator of circulating oxidized LDL was tested by comparing LDL-BDC to results obtained by the immunological autoantibody method. Results: BDC values in LDL isolated by heparin precipitation did not differ from those isolated by sequential ultracentrifugation. While highest amount of diene conjugation was found in LDL (40% of that in serum), substantial amounts were also found in VLDL (31%) and HDL (25%). When analyzed in the same samples, assays for the titer of autoantibodies against oxidized LDL and LDL-BDC were found to show good correlation (r 5 0.57, p 5 0.001, n 5 29). Conclusions: These results, together with thus far conducted studies on clinical applicability of the method, indicate that LDLBDC is a promising candidate in search for a method for estimation of LDL oxidation in vivo. Copyright © 1998 The Canadian Society of Clinical Chemists
KEY WORDS: low-density lipoprotein; oxidation; atherosclerosis; lipid peroxidation; in vivo; human serum.
Introduction everal lines of evidence now indicate that oxidation of low-density lipoprotein (LDL) is a key step in atherogenesis. Oxidation alters biological properties of LDL, and these alterations lead to the development of atherosclerotic lesions (1,2). Thus far, the data on LDL oxidation come largely from studies
S
where kinetics of the oxidation of isolated LDL has been analyzed in vitro. In these studies one of the most widely used methods for monitoring LDL oxidation has been the spectrophotometric determination of the formation of conjugated dienes (3). At present, there is a paucity of methods for measurement of in vivo LDL oxidation (for review, see ref. 3). Of the direct methods currently available for estimation of in vivo LDL oxidation, the preferred one has been the immunological method, based on the use of autoantibodies to epitopes on oxidized LDL (3). The current methodology is rather complex and poorly applicable for clinical purposes. Therefore, we have developed a fast assay for LDL oxidation products, which is based on (a) precipitation of LDL with buffered heparin, and (b) determination of baseline levels of conjugated dienes in lipids extracted from LDL (4). For isolation of the plasma lipoprotein fractions, sequential ultracentrifugation is regarded as the reference method. We have, therefore, performed a detailed comparison between the ultracentrifugation and heparin precipitation methods in order to see whether isolation of LDL by precipitation affects the level of baseline diene conjugation (BDC) in LDL lipids. In addition, the validity of LDL-BDC as an indicator of circulating oxidized LDL was tested by comparing results obtained by this method to those obtained by the immunological autoantibody method. Methods
Correspondence: Dr. Markku Ahotupa, University of Turku, MCA Research Laboratory, Department of Physiology, Biocity, Tykistokatu 6B, FIN-20520 Turku, Finland. Manuscript received November 10, 1997; revised and accepted March 16, 1998. CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
APPARATUS In case of diene conjugation analyses, spectrophotometric analyses were performed with PerkinElmer Lambda 2 spectrometer (Perkin-Elmer Corp., 257
AHOTUPA
Norwalk, CT, USA), in case of the immunological analysis with the microplate reader (Multiscan MCC/340, Labsystems GmbH, Munich, Germany). The equipment were connected to personal computers with special programs for the assays. For preparative ultracentrifugation we used a Kontron TGA-65 ultracentrifuge with TST 60.4 swing-out rotors.
ET AL.
ULTRACENTRIFUGAL
LDL was isolated by sequential ultracentrifugation from human serum containing 1 mg/mL EDTA as described (6). Determination of LDL oxidation products and antioxidant potential was carried out immediately after isolation. ANALYSIS
OF OXIDIZED
CONJUGATION IN
REAGENTS
ANALYSIS
LDL
LDL LIPIDS
BY BASELINE DIENE
(LDL-BDC)
Reagent kits for cholesterol (CHOD-PAP method) were from Boehringer Mannheim (Mannheim, Germany). Heparin was obtained from Loevus Kemiska Fabrik (Ballerup, Denmark), chloroform (p.a.), methanol (p.a.), and cyclohexan (Uvasol) from E. Merck (Darmstadt, Germany). All other reagents were from Sigma Chemicals Co. (St. Louis, MO, USA).
For estimation of LDL oxidation by the baseline level of diene conjugation in LDL lipids, the following procedure was used (4): lipids were extracted from LDL samples (100 mL) by chloroform-methanol (2:1), dried under nitrogen, then redissolved in cyclohexane, and analyzed spectrophotometrically at 234 nm. Absorbance units (difference A234–A300) were converted to molar units using the molar extinction coefficient 2.95 3 104 M21 cm21.
SUBJECTS
ANALYSIS OXIDIZED
For the study comparing the various LDL isolation techniques, a pooled serum obtained from 120 men and women of various ages (Turku City Hospital), was used. Healthy men (n 5 29), aged 21– 44 years, volunteered for the study on correspondence between the two different measures of oxidized LDL. These subjects were in general good health, and the study was done in accordance with the Helsinki Declaration of 1975, as revised in 1983. BLOOD
COLLECTION
Blood samples were obtained by venipuncture after an overnight fast. The blood samples were collected into 10 mL Vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA), allowed to stand for 30 min at room temperature (protected from UV-light), and the serum was separated from cells by centrifugation at 3000 g for 15 min. PRECIPITATION
OF LOW DENSITY LIPOPROTEINS
Serum low density lipoproteins were precipitated by buffered heparin as described (5). The precipitation buffer consisted of 0.064 M trisodium citrate adjusted to pH 5.05 with 5 N HCl, and contained 50,000 IU/L heparin. Before precipitation of low density lipoproteins, serum samples (to which 1 mg/mL of EDTA were added) and precipitation reagents were allowed to equilibrate to room temperature. 1 mL of the sample was added to 7 mL of the heparin-citrate buffer. After mixing with a Vortex mixer the suspension was allowed to stand for 10 min at room temperature. The insoluble lipoproteins were then sedimented by centrifugation at 1000 g for 10 min. The pellet was resuspended in 1 mL of 0.1 M Na-phosphate buffer, pH 8.0, containing 0.9% of NaCl. 258
OF OXIDIZED
LDL
BY AUTOANTIBODIES AGAINST
LDL
Autoantibody titers of anti-oxidized LDL were measured by enzyme-linked immunosorbent assay method (7,8). Antigens for this assay included native LDL, protected against oxidation by 0.27 mM edetic acid and butylated hydroxytoluene (BHT) in phosphate buffered saline (PBS, 10 mM sodium phosphate, pH 7.2), and oxidized LDL (obtained after 18 h oxidation with 2 mM CuSO4, and prepared from the pooled plasma of ten donors). The wells were incubated (coated) with 50 mL of native and oxidized LDL antigen (5 mg/mL) in PBS for 16 h at 4° C. After removal of the unbound antigen and washing of the wells (with PBS containing 0.05% Tween 20, then with distilled water), the remaining nonspecific binding sites were saturated using 2% bovine serum albumin in PBS. The wells were washed, and 50 mL of serum sample (diluted to 1:20 and 1:50) were added to wells coated with native LDL and oxidized LDL, and incubated over night at 4° C. After incubation, the wells were aspirated and washed, before an appropriate IgG-peroxidase conjugated rabbit antihuman monoclonal antibody (Organon, USA, no. 55220 Cappel, diluted to 1:4000 in 0.27 mM PBS, 20 mM edetic acid, 1% BHT, bovine serum albumin0.05% Tween) was added to each well (50 mL). After incubation (for 4 h at 4° C) the unbound material from the wells were aspirated and wells washed. After this, 50 mL of freshly prepared substrate (0.4 mg/mL o-phenylenediamine and 0.045% H2O2 in 100 mM acetate buffer, pH 5.4) was added and incubated for 5 min at room temperature. The enzyme reaction was terminated by addition of 50 mL of 2 M H2SO4. The optical density was then measured spectrophotometrically at 492 nm. To calculate the antibody titer, we used the ratio of the corresponding spectrophotometric reading of antioxidized LDL and the anti-native LDL wells from the same serum sample. Using this approach, the CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
CIRCULATING OXIDIZED LDL
TABLE 1 Baseline Diene Conjugation (BDC) in Serum and Lipoprotein Fractions Isolated by Sequential Ultracentrifugation (UC) or Heparin Precipitation (HEP)
Serum
UC-VLDL
UC-LDL
UC-HDL
HEP-LDL
Poola
BDCb
I II III IV Total mean I II III IV Total mean I II III IV Total mean I II III IV Total mean I II III IV Total mean
71.8 6 2.7 68.1 6 1.1 57.7 6 5.9 68.3 6 1.4 66.5 6 6.1 26.4 6 6.4 20.9 6 3.3 16.3 6 2.4 18.0 6 3.2 20.4 6 4.4 25.1 6 2.8 26.3 6 2.9 23.4 6 1.1 30.8 6 1.8 26.4 6 3.2 12.6 6 1.9 20.3 6 2.3 15.3 6 2.2 19.0 6 1.5 16.8 6 3.5 25.1 6 0.9 33.3 6 13.6 24.6 6 0.5 26.7 6 1.4 27.4 6 4.0
a
LDL isolation by ultracentrifugation and heparin precipitation was repeated 4 times, on separate days and with freshly prepared serum pools. The Roman numerals refer to the different isolation times and serum pools. “Total mean” is the mean 6 SD of means of the four different serum pools. b Results for the various isolation times/serum pools are given as mmol/L, and are mean 6 SD from 6 different determinations (from each serum pool, 6 separate LDL samples were isolated).
spectrophotometric readings of antinative LDL wells represent the corresponding blanks of antioxidized LDL wells and reduce the possible detection of false-positive values. Results Table 1 shows data on BDC levels in various lipoprotein fractions, and also comparison between the different techniques for LDL isolation. The lipoprotein fractions contained practically all (96%) of the BDC in serum; highest BDC levels were found in LDL (40%), followed by VLDL (31%) and HDL (25%). Importantly, BDC values in LDL isolated by the heparin precipitation method did not differ from those in LDL isolated by sequential ultracentrifugation (statistical significance calculated by Student’s t-test) (Table 1). A study on the correspondence between the LDLBDC method and the immunological autoantibody -method for oxidized LDL was performed with 29 healthy middle-aged men. The results (Figure 1) CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
show that these two methods are in agreement with each other. Discussion Mechanisms by which oxidized LDL contributes to various stages of the atherotic process have been worked out by studies with experimental animals or in vitro models. Yet, the data confirming the key role of oxidized LDL in development of atherosclerosis in humans in vivo has remained limited (9). One reason for the scantiness of studies on humans has been the lack of methods for direct measurement of oxidized LDL, applicable for the necessary large-scale epidemiological studies. Due to the heterogenous nature of the chemistry of LDL oxidation, proper determination of “oxidized LDL” is problematic. LDL oxidation may involve various constituents of LDL (e.g., polyunsaturated fatty acids, cholesterol, protein), and each of these can give rise to a number of different kinds of oxidation products. Immunological methods for determination of oxidized LDL are based on the use of antibodies generated either in vivo or raised against in vitro oxidatively damaged LDL. Knowing the multiplicity of LDL oxidation products, it is inevitable that several possible antigens appear diminishing specificity of the assays. Indeed, it was reported that antibodies prepared to identify oxidized LDL recognize also epitopes on proteins other than LDL (10). Use of a valid indicator of LDL oxidation is, of course, fundamental for the study on oxidized LDL in atherosclerosis. After introduction of the method by Esterbauer et al. (11), measurement of diene conjugation has become the most popular method to monitor oxidation of LDL in vitro (cf. 3). Rearrangement of double bonds in polyunsaturated fatty acids, i.e., the formation of conjugated dienes, is an early event of lipid peroxidation taking place soon after initiation of the chain reaction (12), and the oxidation-induced increase of diene conjugation in LDL lipids is well documented (13). Our method for the direct measurement of oxidized LDL (LDL-BDC) measures baseline level of diene conjugation in circulating LDL. The assay is fast and simple to perform, shows good reproducibility and linearity, and can thus be applied to clinical purposes (4). The LDL-BDC assay is based on spectrophotometric analysis of LDL lipids. Hence, it is possible that lipophilic substances of plasma that are transported with the LDL fraction and absorb light at 234 nm may potentially interfere with the assay. One such candidate are products or intermediates of the cyclooxygenase pathway. Inhibition of this pathway by aspirin does not, however, affect the LDL-BDC level (unpublished results), suggesting lack of interference by these compounds. Similarly vitamin E, which may influence the BDC measurements done in serum, does not normally (when serum a-tocopherol concentration is less than 200 mmol/L) interfere measurement of BDC in LDL fraction (unpublished results). 259
AHOTUPA
ET AL.
Figure 1 — Scatterplot showing the relationship between LDL baseline diene conjugation (BDC) and autoantibodies against oxidized LDL (ox-LDL), expressed as optical density units. Pearson’s correlation coefficient r 5 0.567, p 5 0.0013.
For reliability of the LDL-BDC assay and, more generally, for all the assays used for estimation of oxidative modifications of endogenous molecules, it is essential that sampling or sample workup procedures do not result in oxidation. We have found that addition of antioxidants to blood or serum (in addition to the routinely added EDTA) samples has no effect on LDL-BDC (unpublished results). Additionally, in the present study we show that BDC values measured in heparin precipitated LDL are not different from those measured in LDL isolated by the conventional ultracentrifugation method. Thus, sample preparation by the fast precipitation method, which multiplies efficacy of the assay, does not affect the resulting BDC value. In addition, these experiments showed that while most of the serum BDC (40%) is associated with the LDL fraction, substantial amounts of diene conjugation are also found in VLDL (31%) and HDL (25%) fractions. Distribution of conjugated dienes between the lipoprotein fractions is similar to what was reported in various early studies for thiobarbituric acid reactive substances (TBARS): of the TBARS associated with lipoprotein fractions 42 to 47% is found in LDL, 22 to 26% in VLDL and 22 to 34% in HDL (data collected in ref. 13). Yet, although a great portion of serum BDC is associated with the LDL fraction, we have found that serum BDC may be changed (e.g., due to oxidative stress induced by physical activity) without a noticeable change in LDL-BDC (14). In the present study, a positive correlation was found to exist between the LDL-BDC value and the autoantibodies against epitopes on oxidized LDL, even though for a more thorough comparison more data points would have been needed. Yet, this finding supports the view that diene conjugation is a 260
useful index of LDL oxidation, and that the usefulness is not limited to in vitro studies but does also apply to the measurement of oxidized LDL in vivo. Thus far, clinical applicability of the LDL-BDC method has been tested by selected small-scale studies among volunteers and patients with life style dependent or disease-dependent factors known to affect the risk of atherosclerosis and coronary heart disease. In these studies we have, e.g., found that middle-aged men (n 5 31) who are actively participating in endurance training have distinctly lower (37%) LDL-BDC values than age- and weightmatched controls with similar socioeconomic background, and dietary and smoking habits (15). Moreover, controlled reduction of body weight (by 13%) among obese premenopausal women (n 5 82) decreased significantly LDL-BDC levels (total LDLBDC by 40%; LDL-BDC/total LDL cholesterol ratio by 32%) (16). In an unpublished study, we found a positive correlation between LDL-BDC and the intimal-medial thickness in the common carotid artery. Studies with twins and their families showed that inherited factors contribute to interindividual variability of the LDL-BDC (17). We have also found that short-term antioxidant intervention does not affect LDL-BDC in young and middle-aged healthy volunteers, even though the antioxidant potential of LDL is substantially increased (4,18). Taken together, the LDL-BDC method is fast and easy to perform with instrumentation generally available in clinical laboratories. The present knowledge on validity and applicability of the method clearly indicate that LDL-BDC is among the most promising candidates in search for methods for the direct measurement of LDL oxidation in vivo. CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
CIRCULATING OXIDIZED LDL
Acknowledgements This work was supported by grants from Juho Vainio Foundation, Finnish Heart Association, Foundation of the University of Turku, Yrjo¨ Jahnsson Foundation, The Medical Foundation of the Tampere University Hospital, The Finnish Foundation of Cardiovascular Research and The Elli and Elvi Oksanen Fund under the Pirkanmaa Cultural Foundation in the auspices of the Finnish Cultural Foundation.
References 1. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med 1996; 20: 707–27. 2. Witztum JL, Stein D. Role of oxidized LDL in atherogenesis. J Clin Invest 1991; 88: 1785–92. 3. Jialal I, Devaraj S. Low-density lipoprotein oxidation, antioxidants, and atherosclerosis: a clinical biochemistry perspective. Clin Chem 1996; 42: 498 –506. 4. Ahotupa M, Ruutu M, Ma¨ntyla¨ E. Simple methods of quantifying oxidation products and antioxidant potential of low density lipoproteins. Clin Biochem 1996; 29: 139 – 44. 5. Wieland H, Seidel D. A simple specific method for precipitation of low density lipoproteins. J Lipid Res 1983; 24: 904 –9. 6. Schumaker VN, Puppione DL. Sequential flotation ultracentrifugation. Methods Enzymol 1986; 128: 155– 68. 7. Uusitupa MI, Niskanen L, Luoma J, et al. Autoantibodies against oxidized LDL do not predict atherosclerotic vascular disease in non-insulin-dependent diabetes mellitus. Arterioscler Thromb Vasc Biol 1996; 16: 1236 – 42. 8. Heitzer T, Yla¨-Herttuala S, Luoma J, et al. Cigarette smoking potentiates endothelial dysfunction of forearm resistance vessels in patients with hypercholesterolemia. Role of oxidized LDL. Circulation 1996; 93: 1346 –53. 9. Yla¨-Herttuala S, Rosenfield ME, Parthasarathy S, et
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10.
11.
12.
13.
14.
15.
16.
17.
18.
al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 1989; 84: 1086 –95. O’Brien KD, Alpers CE, Hokanson JE, Wang S, Chait A. Oxidation-specific epitopes in human coronary atherosclerosis are not limited to oxidized low-density lipoprotein. Circulation 1996; 94: 1216 –25. Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun 1989; 6: 67–75. Kappus H. Lipid peroxidation: mechanisms, analysis, enzymology and biological relevance. In: Sies H, Ed. Oxidative stress. London: Academic Press, 1985: 273– 310. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med 1992; 13: 341–90. Vasankari TJ, Kujala U, Vasankari TM, Vuorimaa T, Ahotupa M. Effects of acute prolonged exercise on serum and LDL oxidation antioxidant defences. Free Rad Biol Med 1997; 22: 509 –13. Kujala UM, Ahotupa M, Vasankari T, Kaprio J, Tikkanen MJ. Low LDL oxidation in veteran endurance athletes. Scand J Med Sci Sports 1996; 6: 303– 8. Vasankari T, Fogelholm M, Oja P, Vuori I, Ahotupa M. Effect of weight reduction on LDL oxidation (abstract). In: Va¨isa¨nen S, Suomela-Markkanen T, Rankinen T, Eds. International 14th Puijo Symposium “Physical Activity, Diet and Cardiovascular Diseases A Fresh Look Beyond Old Facts, Publications of the University of Kuopio, Ser D Medicine 101, Kuopio, Finland, 1996, p. 37. Kujala UM, Ahotupa M, Vasankari T, Kaprio J, Tikkanen MJ. Familial aggregation of LDL oxidation. Scand J Clin Lab Invest 1997; 57: 141– 6. Vasankari TJ, Kujala UM, Vasankari TM, Vuorimaa T, Ahotupa M. Increased serum LDL antioxidant potential after antioxidant supplementation in endurance athletes. Am J Clin Nutr 1997; 65: 1052–56.
261
PII S0009-9120(98)00018-6
Baseline Diene Conjugation in LDL Lipids as a Direct Measure of In Vivo LDL Oxidation MARKKU AHOTUPA,1 JUKKA MARNIEMI,2 TERHO LEHTIMA¨KI,3 KATI TALVINEN,1 OLLI T. RAITAKARI,4 TOMMI VASANKARI,1 JORMA VIIKARI,5 JUKKA LUOMA,6 and SEPPO YLA¨-HERTTUALA6 1
MCA Research Laboratory, Department of Physiology, University of Turku, Turku, Finland, Research and Development Centre, Social Insurance Institution, Turku, Finland, 3Department of Clinical Chemistry, University Hospital of Tampere and Department of Medical Biochemistry, Medical School, University of Tampere, Tampere, Finland, 4Department of Clinical Physiology, University Hospital of Turku, Turku, Finland, 5Department of Internal Medicine, University Hospital of Turku, Turku, Finland, and 6A. I. Virtanen Institute, Kuopio, Finland 2
Objectives: To additionally test validity of the recently developed method (LDL baseline diene conjugation, LDL-BDC) for determination of circulating oxidized LDL. Design and methods: A detailed comparison between the ultracentrifugation and heparin precipitation methods for LDL isolation was performed to test suitability of the fast precipitation method. Validity of LDL-BDC as an indicator of circulating oxidized LDL was tested by comparing LDL-BDC to results obtained by the immunological autoantibody method. Results: BDC values in LDL isolated by heparin precipitation did not differ from those isolated by sequential ultracentrifugation. While highest amount of diene conjugation was found in LDL (40% of that in serum), substantial amounts were also found in VLDL (31%) and HDL (25%). When analyzed in the same samples, assays for the titer of autoantibodies against oxidized LDL and LDL-BDC were found to show good correlation (r 5 0.57, p 5 0.001, n 5 29). Conclusions: These results, together with thus far conducted studies on clinical applicability of the method, indicate that LDLBDC is a promising candidate in search for a method for estimation of LDL oxidation in vivo. Copyright © 1998 The Canadian Society of Clinical Chemists
KEY WORDS: low-density lipoprotein; oxidation; atherosclerosis; lipid peroxidation; in vivo; human serum.
Introduction everal lines of evidence now indicate that oxidation of low-density lipoprotein (LDL) is a key step in atherogenesis. Oxidation alters biological properties of LDL, and these alterations lead to the development of atherosclerotic lesions (1,2). Thus far, the data on LDL oxidation come largely from studies
S
where kinetics of the oxidation of isolated LDL has been analyzed in vitro. In these studies one of the most widely used methods for monitoring LDL oxidation has been the spectrophotometric determination of the formation of conjugated dienes (3). At present, there is a paucity of methods for measurement of in vivo LDL oxidation (for review, see ref. 3). Of the direct methods currently available for estimation of in vivo LDL oxidation, the preferred one has been the immunological method, based on the use of autoantibodies to epitopes on oxidized LDL (3). The current methodology is rather complex and poorly applicable for clinical purposes. Therefore, we have developed a fast assay for LDL oxidation products, which is based on (a) precipitation of LDL with buffered heparin, and (b) determination of baseline levels of conjugated dienes in lipids extracted from LDL (4). For isolation of the plasma lipoprotein fractions, sequential ultracentrifugation is regarded as the reference method. We have, therefore, performed a detailed comparison between the ultracentrifugation and heparin precipitation methods in order to see whether isolation of LDL by precipitation affects the level of baseline diene conjugation (BDC) in LDL lipids. In addition, the validity of LDL-BDC as an indicator of circulating oxidized LDL was tested by comparing results obtained by this method to those obtained by the immunological autoantibody method. Methods
Correspondence: Dr. Markku Ahotupa, University of Turku, MCA Research Laboratory, Department of Physiology, Biocity, Tykistokatu 6B, FIN-20520 Turku, Finland. Manuscript received November 10, 1997; revised and accepted March 16, 1998. CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
APPARATUS In case of diene conjugation analyses, spectrophotometric analyses were performed with PerkinElmer Lambda 2 spectrometer (Perkin-Elmer Corp., 257
AHOTUPA
Norwalk, CT, USA), in case of the immunological analysis with the microplate reader (Multiscan MCC/340, Labsystems GmbH, Munich, Germany). The equipment were connected to personal computers with special programs for the assays. For preparative ultracentrifugation we used a Kontron TGA-65 ultracentrifuge with TST 60.4 swing-out rotors.
ET AL.
ULTRACENTRIFUGAL
LDL was isolated by sequential ultracentrifugation from human serum containing 1 mg/mL EDTA as described (6). Determination of LDL oxidation products and antioxidant potential was carried out immediately after isolation. ANALYSIS
OF OXIDIZED
CONJUGATION IN
REAGENTS
ANALYSIS
LDL
LDL LIPIDS
BY BASELINE DIENE
(LDL-BDC)
Reagent kits for cholesterol (CHOD-PAP method) were from Boehringer Mannheim (Mannheim, Germany). Heparin was obtained from Loevus Kemiska Fabrik (Ballerup, Denmark), chloroform (p.a.), methanol (p.a.), and cyclohexan (Uvasol) from E. Merck (Darmstadt, Germany). All other reagents were from Sigma Chemicals Co. (St. Louis, MO, USA).
For estimation of LDL oxidation by the baseline level of diene conjugation in LDL lipids, the following procedure was used (4): lipids were extracted from LDL samples (100 mL) by chloroform-methanol (2:1), dried under nitrogen, then redissolved in cyclohexane, and analyzed spectrophotometrically at 234 nm. Absorbance units (difference A234–A300) were converted to molar units using the molar extinction coefficient 2.95 3 104 M21 cm21.
SUBJECTS
ANALYSIS OXIDIZED
For the study comparing the various LDL isolation techniques, a pooled serum obtained from 120 men and women of various ages (Turku City Hospital), was used. Healthy men (n 5 29), aged 21– 44 years, volunteered for the study on correspondence between the two different measures of oxidized LDL. These subjects were in general good health, and the study was done in accordance with the Helsinki Declaration of 1975, as revised in 1983. BLOOD
COLLECTION
Blood samples were obtained by venipuncture after an overnight fast. The blood samples were collected into 10 mL Vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA), allowed to stand for 30 min at room temperature (protected from UV-light), and the serum was separated from cells by centrifugation at 3000 g for 15 min. PRECIPITATION
OF LOW DENSITY LIPOPROTEINS
Serum low density lipoproteins were precipitated by buffered heparin as described (5). The precipitation buffer consisted of 0.064 M trisodium citrate adjusted to pH 5.05 with 5 N HCl, and contained 50,000 IU/L heparin. Before precipitation of low density lipoproteins, serum samples (to which 1 mg/mL of EDTA were added) and precipitation reagents were allowed to equilibrate to room temperature. 1 mL of the sample was added to 7 mL of the heparin-citrate buffer. After mixing with a Vortex mixer the suspension was allowed to stand for 10 min at room temperature. The insoluble lipoproteins were then sedimented by centrifugation at 1000 g for 10 min. The pellet was resuspended in 1 mL of 0.1 M Na-phosphate buffer, pH 8.0, containing 0.9% of NaCl. 258
OF OXIDIZED
LDL
BY AUTOANTIBODIES AGAINST
LDL
Autoantibody titers of anti-oxidized LDL were measured by enzyme-linked immunosorbent assay method (7,8). Antigens for this assay included native LDL, protected against oxidation by 0.27 mM edetic acid and butylated hydroxytoluene (BHT) in phosphate buffered saline (PBS, 10 mM sodium phosphate, pH 7.2), and oxidized LDL (obtained after 18 h oxidation with 2 mM CuSO4, and prepared from the pooled plasma of ten donors). The wells were incubated (coated) with 50 mL of native and oxidized LDL antigen (5 mg/mL) in PBS for 16 h at 4° C. After removal of the unbound antigen and washing of the wells (with PBS containing 0.05% Tween 20, then with distilled water), the remaining nonspecific binding sites were saturated using 2% bovine serum albumin in PBS. The wells were washed, and 50 mL of serum sample (diluted to 1:20 and 1:50) were added to wells coated with native LDL and oxidized LDL, and incubated over night at 4° C. After incubation, the wells were aspirated and washed, before an appropriate IgG-peroxidase conjugated rabbit antihuman monoclonal antibody (Organon, USA, no. 55220 Cappel, diluted to 1:4000 in 0.27 mM PBS, 20 mM edetic acid, 1% BHT, bovine serum albumin0.05% Tween) was added to each well (50 mL). After incubation (for 4 h at 4° C) the unbound material from the wells were aspirated and wells washed. After this, 50 mL of freshly prepared substrate (0.4 mg/mL o-phenylenediamine and 0.045% H2O2 in 100 mM acetate buffer, pH 5.4) was added and incubated for 5 min at room temperature. The enzyme reaction was terminated by addition of 50 mL of 2 M H2SO4. The optical density was then measured spectrophotometrically at 492 nm. To calculate the antibody titer, we used the ratio of the corresponding spectrophotometric reading of antioxidized LDL and the anti-native LDL wells from the same serum sample. Using this approach, the CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
CIRCULATING OXIDIZED LDL
TABLE 1 Baseline Diene Conjugation (BDC) in Serum and Lipoprotein Fractions Isolated by Sequential Ultracentrifugation (UC) or Heparin Precipitation (HEP)
Serum
UC-VLDL
UC-LDL
UC-HDL
HEP-LDL
Poola
BDCb
I II III IV Total mean I II III IV Total mean I II III IV Total mean I II III IV Total mean I II III IV Total mean
71.8 6 2.7 68.1 6 1.1 57.7 6 5.9 68.3 6 1.4 66.5 6 6.1 26.4 6 6.4 20.9 6 3.3 16.3 6 2.4 18.0 6 3.2 20.4 6 4.4 25.1 6 2.8 26.3 6 2.9 23.4 6 1.1 30.8 6 1.8 26.4 6 3.2 12.6 6 1.9 20.3 6 2.3 15.3 6 2.2 19.0 6 1.5 16.8 6 3.5 25.1 6 0.9 33.3 6 13.6 24.6 6 0.5 26.7 6 1.4 27.4 6 4.0
a
LDL isolation by ultracentrifugation and heparin precipitation was repeated 4 times, on separate days and with freshly prepared serum pools. The Roman numerals refer to the different isolation times and serum pools. “Total mean” is the mean 6 SD of means of the four different serum pools. b Results for the various isolation times/serum pools are given as mmol/L, and are mean 6 SD from 6 different determinations (from each serum pool, 6 separate LDL samples were isolated).
spectrophotometric readings of antinative LDL wells represent the corresponding blanks of antioxidized LDL wells and reduce the possible detection of false-positive values. Results Table 1 shows data on BDC levels in various lipoprotein fractions, and also comparison between the different techniques for LDL isolation. The lipoprotein fractions contained practically all (96%) of the BDC in serum; highest BDC levels were found in LDL (40%), followed by VLDL (31%) and HDL (25%). Importantly, BDC values in LDL isolated by the heparin precipitation method did not differ from those in LDL isolated by sequential ultracentrifugation (statistical significance calculated by Student’s t-test) (Table 1). A study on the correspondence between the LDLBDC method and the immunological autoantibody -method for oxidized LDL was performed with 29 healthy middle-aged men. The results (Figure 1) CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
show that these two methods are in agreement with each other. Discussion Mechanisms by which oxidized LDL contributes to various stages of the atherotic process have been worked out by studies with experimental animals or in vitro models. Yet, the data confirming the key role of oxidized LDL in development of atherosclerosis in humans in vivo has remained limited (9). One reason for the scantiness of studies on humans has been the lack of methods for direct measurement of oxidized LDL, applicable for the necessary large-scale epidemiological studies. Due to the heterogenous nature of the chemistry of LDL oxidation, proper determination of “oxidized LDL” is problematic. LDL oxidation may involve various constituents of LDL (e.g., polyunsaturated fatty acids, cholesterol, protein), and each of these can give rise to a number of different kinds of oxidation products. Immunological methods for determination of oxidized LDL are based on the use of antibodies generated either in vivo or raised against in vitro oxidatively damaged LDL. Knowing the multiplicity of LDL oxidation products, it is inevitable that several possible antigens appear diminishing specificity of the assays. Indeed, it was reported that antibodies prepared to identify oxidized LDL recognize also epitopes on proteins other than LDL (10). Use of a valid indicator of LDL oxidation is, of course, fundamental for the study on oxidized LDL in atherosclerosis. After introduction of the method by Esterbauer et al. (11), measurement of diene conjugation has become the most popular method to monitor oxidation of LDL in vitro (cf. 3). Rearrangement of double bonds in polyunsaturated fatty acids, i.e., the formation of conjugated dienes, is an early event of lipid peroxidation taking place soon after initiation of the chain reaction (12), and the oxidation-induced increase of diene conjugation in LDL lipids is well documented (13). Our method for the direct measurement of oxidized LDL (LDL-BDC) measures baseline level of diene conjugation in circulating LDL. The assay is fast and simple to perform, shows good reproducibility and linearity, and can thus be applied to clinical purposes (4). The LDL-BDC assay is based on spectrophotometric analysis of LDL lipids. Hence, it is possible that lipophilic substances of plasma that are transported with the LDL fraction and absorb light at 234 nm may potentially interfere with the assay. One such candidate are products or intermediates of the cyclooxygenase pathway. Inhibition of this pathway by aspirin does not, however, affect the LDL-BDC level (unpublished results), suggesting lack of interference by these compounds. Similarly vitamin E, which may influence the BDC measurements done in serum, does not normally (when serum a-tocopherol concentration is less than 200 mmol/L) interfere measurement of BDC in LDL fraction (unpublished results). 259
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Figure 1 — Scatterplot showing the relationship between LDL baseline diene conjugation (BDC) and autoantibodies against oxidized LDL (ox-LDL), expressed as optical density units. Pearson’s correlation coefficient r 5 0.567, p 5 0.0013.
For reliability of the LDL-BDC assay and, more generally, for all the assays used for estimation of oxidative modifications of endogenous molecules, it is essential that sampling or sample workup procedures do not result in oxidation. We have found that addition of antioxidants to blood or serum (in addition to the routinely added EDTA) samples has no effect on LDL-BDC (unpublished results). Additionally, in the present study we show that BDC values measured in heparin precipitated LDL are not different from those measured in LDL isolated by the conventional ultracentrifugation method. Thus, sample preparation by the fast precipitation method, which multiplies efficacy of the assay, does not affect the resulting BDC value. In addition, these experiments showed that while most of the serum BDC (40%) is associated with the LDL fraction, substantial amounts of diene conjugation are also found in VLDL (31%) and HDL (25%) fractions. Distribution of conjugated dienes between the lipoprotein fractions is similar to what was reported in various early studies for thiobarbituric acid reactive substances (TBARS): of the TBARS associated with lipoprotein fractions 42 to 47% is found in LDL, 22 to 26% in VLDL and 22 to 34% in HDL (data collected in ref. 13). Yet, although a great portion of serum BDC is associated with the LDL fraction, we have found that serum BDC may be changed (e.g., due to oxidative stress induced by physical activity) without a noticeable change in LDL-BDC (14). In the present study, a positive correlation was found to exist between the LDL-BDC value and the autoantibodies against epitopes on oxidized LDL, even though for a more thorough comparison more data points would have been needed. Yet, this finding supports the view that diene conjugation is a 260
useful index of LDL oxidation, and that the usefulness is not limited to in vitro studies but does also apply to the measurement of oxidized LDL in vivo. Thus far, clinical applicability of the LDL-BDC method has been tested by selected small-scale studies among volunteers and patients with life style dependent or disease-dependent factors known to affect the risk of atherosclerosis and coronary heart disease. In these studies we have, e.g., found that middle-aged men (n 5 31) who are actively participating in endurance training have distinctly lower (37%) LDL-BDC values than age- and weightmatched controls with similar socioeconomic background, and dietary and smoking habits (15). Moreover, controlled reduction of body weight (by 13%) among obese premenopausal women (n 5 82) decreased significantly LDL-BDC levels (total LDLBDC by 40%; LDL-BDC/total LDL cholesterol ratio by 32%) (16). In an unpublished study, we found a positive correlation between LDL-BDC and the intimal-medial thickness in the common carotid artery. Studies with twins and their families showed that inherited factors contribute to interindividual variability of the LDL-BDC (17). We have also found that short-term antioxidant intervention does not affect LDL-BDC in young and middle-aged healthy volunteers, even though the antioxidant potential of LDL is substantially increased (4,18). Taken together, the LDL-BDC method is fast and easy to perform with instrumentation generally available in clinical laboratories. The present knowledge on validity and applicability of the method clearly indicate that LDL-BDC is among the most promising candidates in search for methods for the direct measurement of LDL oxidation in vivo. CLINICAL BIOCHEMISTRY, VOLUME 31, JUNE 1998
CIRCULATING OXIDIZED LDL
Acknowledgements This work was supported by grants from Juho Vainio Foundation, Finnish Heart Association, Foundation of the University of Turku, Yrjo¨ Jahnsson Foundation, The Medical Foundation of the Tampere University Hospital, The Finnish Foundation of Cardiovascular Research and The Elli and Elvi Oksanen Fund under the Pirkanmaa Cultural Foundation in the auspices of the Finnish Cultural Foundation.
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