Sobenin IA, Tertov VV, Orekhov AN
J Lipid Res 1998 Nov 39(11):2293-2299.

Abstract

Sialic acid level in blood plasma and circulating glycoproteins is considered to be a marker for a number of pathologic conditions, including atherosclerosis, cancer, etc. The precise measurement of sialic acid level is an important laboratory procedure to allow correct interpretation of results. Colorimetric methods commonly used for the measurement of sialic acid are not highly specific, as interfering substances may alter the results. Among these, malondialdehyde and other aldehydes play the decisive role. In the circulation, aldehydes are commonly produced during lipid peroxidation in the lipid core of lipoprotein particles, especially low density lipoprotein (LDL). To establish the impairment to the sialic acid determination in LDL introduced by interfering substances, the optimized assay based on Warren's traditional method was developed and tested in 606 LDL samples. The optimization implies the comparison of color developed using the standard Warren procedure with that due to contaminating agents, mainly thiobarbituric acid-reactive substances (TBARS). In LDL stored at 4 degreesC, the estimates obtained by the modified procedure were 41.5% or 30.1 nmol/mg lower, on average, compared to the standard procedure (n = 45, P < 0.0001). Even in LDL stored at -70 degreesC, sialic acid estimates obtained by the modified procedure were 6.6% or 3.6 nmol/mg lower, on average, compared to the standard measurement (n = 561, P < 0.005). Thus, the modified procedure avoids significant distortion of the measurement induced by the presence of interfering agents.

Introduction

In the recent years the data on the presence of sialic acid-poor low density lipoprotein (LDL) in human blood are rapidly accumulating. Several years ago it was demonstrated that LDL from patients with angiographically assessed coronary atherosclerosis, unlike LDL from healthy subjects, induced lipid accumulation in cultured human intimal cells, this effect being called atherogenicity (1). At that time the sialic acid content was the only statistically significant difference between LDL from coronary atherosclerotic patients and healthy subjects (2). So, it was proposed that LDL sialic acid level may play a role in LDL atherogenicity, and a number of experimental studies gave a considerable background for such assumption (3-5). The high atherogenic potential of sialic acid-poor LDL lead to the increasing interest to this type of LDL modification, and several studies have been performed trying to assess clinical significance of low LDL sialic acid level (6,7). Therefore, the precise sialic acid measurement in LDL becomes very important for the correct interpretation of the obtained results. At a time, few methods are commonly used for the measurement of sialic acid levels, and the most popular are thiobarbituric acid (TBA) assay by Warren (8) and resorcinol method by Svennerholm (9). These colorimetric methods based on the formation of chromophores are not highly specific, and some interfering substances may crucially alter the results of measurement, that may lead to erroneous conclusions. Among such substances are malondialdehyde produced during lipid peroxidation processes occurring in the lipid core of LDL particle, and fructose released from non-enzymatically glycosylated proteins. To analyze the influence of these substances, we have developed the semi-micro thiobarbituric acid-based assay for sialic acid determination in LDL, that is the Warren's method modification.

 

Materials and Methods

Reagents

N-acetylneuraminic acid (NANA) was purchased from Calbiochem Corp. (La Jolla, CA), d-galactose was from Serva Feinbiochemica (Heidelberg, Germany), d(-)-fructose was from EM Science (Gibbstown, NJ), cyclohexanone was from Fluka AG (Buchs, Switzerland), all other reagents were from Sigma Chemical Co. (St.Louis, MO).

Sialic acid measurement

LDL sialic acid content was measured in 471 subjects during health screening study, as well as in 112 type 1 and type 2 diabetic patients and in 23 hyperlipidemic subjects.

Venous blood for experiments (15 ml) was taken after overnight fasting in plastic tube containing 1 mM ethylenediamine tetraacetic acid (EDTA). Plasma was separated by centrifugation (20 min at 900 g), and LDL (density, 1.025 to 1.063 g/ml) was isolated by fast two-step preparative ultracentrifugation as described earlier (10). LDL preparations were dialyzed against 2,000 vol phosphate buffered saline (PBS), pH 7.4, containing 1 mM EDTA overnight at 4°C, sterilized by filtration (pore size, 0.45 mm) and stored either at -70°C for 1-8 weeks, or at 4°C for 1-5 days prior to sialic acid measurement. LDL protein was measured according to Lowry et al. (11) immediately before sialic acid assay.

Standard aqueous 1 mM solution of N-acetyl neuraminic acid (Calbiochem Corp., La Jolla, CA, Cat.No 110137) was prepared once and stored in aliquots of 100 ml at -20°C; one aliquot was thawed and used for each experiment and stored no more. Standards containing 0-5-10-25 ml in triplicate were taken for the assay. LDL sialic acid content was measured by standard Warren's assay (8) as well as by modified thiobarbituric acid-based method that is described in the following way. LDL preparation aliquots of 100 ml containing 50-120 mg of LDL protein were taken in triplicate in glass tubes ("assay samples") and additional three aliquots were taken for "correction samples". One hundred microliters of 0.2N H2SO4 was added, and the samples were incubated for 1 hour at 80°C for mild acidic hydrolysis. After that, the tubes were cooled at 20°C for 20 min, and 20 ml of periodate reagent (0.2M NaIO4 in 9M H3PO4) was added to the assay samples. The reagent added to correction samples contained no sodium periodate, i.e. it was simply 9M H3PO4. The tubes were vortexed and incubated for 20 min at 20°C temperature. Then 100 ml of m-arsenite reagent (10% NaAsO2 in 0.1N H2SO4 with 0.5 M Na2SO4) was added and tubes were vortexed vigorously until the yellow-brownish coloration appeared and further disappeared entirely. Then 250 ml of thiobarbituric acid reagent (0.6% TBA in 0.5M Na2SO4) was added, the tubes were vortexed and heated in boiling water bath for 15 min, then cooled at 20°C temperature. After this, 1 ml of cyclohexanone was added and the tubes were vortexed vigorously for 10 sec twice and then centrifuged (7 min at 900 g) for phase separation. Optical density of the organic phase was determined at 549 nm in quartz 0.4-ml cuvette with 1cm light path against blank samples in Yanako UO-2000 model spectrophotometer (Bausch & Lomb, USA). Alternative way of measurement was the chromophore extraction into 1 ml of tert-butanol followed by fluorescence measurement at l_ex 544 nm, l_em 584 nm in black 96-well fluororeader plates in Labsystems Fluoroscan II model fluororeader (Labsystems OY, Finland). The readings of correction samples were subtracted from those of assay samples, thus corrected readings were obtained. Further calculations of sialic acid content in the sample were performed according to calibration curve that was exactly linear within the given range.

For sialic acid measurement by resorcinol method (9), LDL preparation aliquots of 100 ml containing 50-120 mg of LDL protein were taken in triplicate in glass tubes and 900 ml of 5% trichloroacetic acid was added. After mild acidic hydrolysis (7 min at boiling water bath) the samples were centrifuged (5 min at 900 g) and 0.5 ml of clear supernatant was transferred to other tubes. Five hundred microliters of resorcinol reagent (0.2% resorcinol in 30% HCl with 0.25 mM CuSO4) was added and the samples were incubated for 15 min at boiling water bath. After this, 1 ml of butylacetate-butanol 85:15 (vol/vol) was added and the tubes were vortexed vigorously for 10 sec twice and then centrifuged (7 min at 900 g) for phase separation. Optical density of the organic phase was determined at 615 nm in quartz 0.4-ml cuvette with 1cm light path against blank samples.

For the assessment of interfering substances effect, d-galactose, d-fructose, d-mannose, d-glucose, d-glucosamine and d-galactosamine, as well as malondialdehyde were tested in each type of assay in various concentrations.

The TBARS level was measured according to Yagi (12).

Statistical methods

Results are reported as mean + S.E.M. Significance of differences was evaluated by one-way ANOVA and two-tailed Student's paired t-test and was assumed for P values <0.05.

Results

The results of the study are presented in (Table 1). In population-based group, the LDL sialic acid level measured by modified Warren's assay was by 6.6%, or 3.6 nmol/mg lower on an average as compared to standard Warren's measurement. However, this small difference was statistically significant. The conditions of sample storage were of great importance. So, the above data were obtained in those LDL preparations that were stored at -70°C for 1 to 8 weeks prior to the date of assay. Contrarily, the limited number of samples from population-based group were stored at 4°C for 1 to 5 days prior to the assay. In these samples, the corrected sialic acid level was by 41.5%, or 30.1 nmol/mg lower on an average as compared estimates obtained from standard procedure (Table 1). Thus, the standard measurement of LDL sialic acid levels in two population-based groups could give a rise to the assumption that mean levels differed significantly (at p<0.0001), but this difference was an artifact, as it disappeared entirely when the measurement was performed in modified manner (p=0.274).

 

In LDL stored at -70°C, the error of sialic acid content determination (the difference in estimates between non-modified and modified assays) did not exceed 10% in 83% samples, and only in 5% samples the error exceeded 30% of the corrected level (Fig.1, panel B). On the opposite, in LDL stored at 4°C, the error did not exceed 10% only in 32% samples, and in 46% cases the error was greater than 30%. Moreover, under such storage conditions, in 9% samples the difference in estimates between non-modified and modified assays was greater than 2-fold (Fig.1, panel A).

Taking into account the significant difference between sialic acid levels measured by Warren's method in LDL stored under different conditions, we have studied the effect of LDL storage duration on the error level in sialic acid determination (Fig.2). In freshly isolated LDL the levels of sialic acid were similar in spite of the standard or modified method used (33.1±0.1 vs. 32.9±0.1 nmol/mg LDL protein, respectively). In LDL stored for one day at +4°C, the estimated levels of sialic acid content already increased significantly and the rise continued up to the 7th day, but the correction used for modified assay yielded the levels close to the initial one up to the 2nd day of storage. However, at longer storage time (beginning from the 3rd day) both methods gave statistically significant excessive estimates, and even the usage of correction for interfering substances failed to yield the levels similar to initial one (Fig.2). It is important to notice that LDL for this experiment was obtained from healthy volunteer and characterized by low content of TBARS. During the storage TBARS content increased significantly, and measured TBARS level corresponded well to the difference in sialic acid estimates between standard and modified assays (r=0.88, p<0.05).. The addition of EDTA but not butylated hydroxytoluene (BHT) to LDL stored at +4°C prevented the increase of sialic acid estimates up to the 4th day of storage, but at the 7th day even EDTA addition could not anticipate the inaccurate results of measurement (Fig.2). To investigate whether oxidation may lead to erroneous sialic acid estimates, in vitro oxidized LDL were subjected to standard and modified procedures of sialic acid measurement (Fig.3). Already after 1 h of in vitro oxidation, standard measurement produced extremely high values of absorbance, whereas modified procedure yielded the values close to initial one up to 8 h of oxidation (Fig.3). Thus, the error in LDL sialic acid measurement by Warren's assay is due to the presence of products of oxidation, mainly TBARS.

In diabetic patients group, the LDL sialic acid level measured by modified Warren's assay was by 15.6%, or 4.9 nmol/mg lower on an average as compared to standard measurement, the difference being statistically significant (Table 1).

The difference between LDL sialic acid content estimated by two methods was very remarkable for the group of hyperlipidemic subjects. The level measured by modified Warren's assay was by 36.0%, or 15.2 nmol/mg lower on an average as compared to standard measurement, the difference being statistically significant (Table 1). It is notable that the samples were stored at -70°C for 1 week till the date of analysis, so the error in sialic acid determination seemed to be due to the initially high level of interfering substances, such as TBARS. Thus, the measurement of LDL sialic acid levels by standard procedure in hyperlipidemic subjects could easily allow the assumption that LDL sialic acid level in this category of patients is significantly higher than in normals (at p<0.0001), but such tendency was not observed when the measurement was performed in modified manner (p=0.71).

For the assessment of the effect of possibly interfering agents on sialic acid measurement, we tested a number of substances, such as d-glucosamine, d-galactosamine, d-mannose, d-glucose, d-galactose, d-fructose and malondialdehyde. Among all substances tested, malondialdehyde was the most effective one forming the colored product upon condensation with thiobarbituric acid. The peak of adsorption was at 446 nm, and molar extinction coefficient for this chromophore was 3.7-fold higher than that for sialic acid, under the same conditions of assay. Among sugars, d-fructose was the only one that could give negligible but measurable effect, molar extinction coefficient being 82-fold lower than that for sialic acid.

Taking into account that Svennerholm's method is widely used for LDL sialic acid determination along with Warren's one, we have tested the possible interference of the same substances on sialic acid measurement by resorcinol assay. It turned out that molar extinction coefficient for sialic acid itself was 8-fold lower than that for Warren's assay, thus demonstrating that resorcinol method was considerably less sensitive than thiobarbituric acid-based one. Malondialdehyde could form a colored product while reacting with resorcinol, and molar extinction coefficient was 7.1-fold higher than that for sialic acid under the same conditions of assay. Most prominent, d-fructose also interfered very effectively giving a rise to too high absorption levels. In fact, fructose formed the yellow-brownish chromophore upon the reaction with resorcinol with absorption peak at 465 nm, but even at wavelength of 615 nm used in the assay for sialic acid measurement, molar extinction coefficient for fructose was only 1.6-fold lower than that for sialic acid, although the absorption for fructose at 615 nm accounted only for 13% of peak value at 465 nm.

Discussion

It has been recently demonstrated that there is in vivo multiple-modified LDL fraction in human blood. This LDL is characterized by numerous alterations in chemical composition and physical properties. The most remarkable one is low protein- and lipid-bound sialic acid content, i.e. this LDL is desialylated one. The LDL sialic acid level, on the whole, reflects the attendance of modified atherogenic LDL in circulation (2,13,14). At present, a number of colorimetric methods for sialic acid measurement are widely used, among them the most popular are Warren's thiobarbituric acid-based assay, Svennerholm's resorcinol method, and resorcinol-periodate method by Jourdian et al. (15). All these methods can hardly be designated as specific, as some widely spread compounds may interfere and alter the results of measurement. Among these are malondialdehyde and fructose, and we have shown in this study that they can yield dramatically increased estimates, erroneously referred to total sialic acid content of LDL.

Theoretically, it is possible to avoid the influence of lipid peroxidation products by preliminary delipidation of LDL samples. However, this approach would give an additional cumbersome step for the procedure, and the obtained levels would characterize not total but protein-bound sialic acid content. The latter may be of great importance, as the decrease in lipid-bound sialic acid content of LDL may be much more prominent than that of protein-bound sialic acid (16). Usually, the level of malondialdehyde in freshly isolated and dialyzed LDL is negligible as compared to that of sialic acid, and in some cases the formation of additional chromophore not related to sialic acid itself may be ignored. However, the inappropriate storage conditions lead to the substantial increase in LDL TBARS content. Moreover, atherosclerotic or diabetic patients may be characterized by already increased MDA levels in LDL (17,18). Taking into account the high molar extinction coefficient for MDA, it would generally be incorrect to disregard the possibility of obtaining erroneously high estimates of sialic acid due to MDA presence.

To clarify the situation with interfering fructose, it may be relevant to discuss briefly the processes of nonenzymatic glycation that occur in all subjects in circulation. During nonenzymatic glycation, sugars act in a time- and concentration-dependent manner, glycating e-amino groups of lysine and N-terminal amino acids. The first glycation product - glycosylamine, or Shiff base - undergoes an Amadori rearrangement over a period of few days and yields the more stable ketoamine, or Amadori product. The latter is a b-ketosylamine existing both in pyranosyl and furanosyl ring forms, and during mild acidic hydrolysis a significant part of Amadori products is released as fructose-resembling furanose derivatives (19). Colorimetric methods do not possess strict specificity and therefore cannot distinguish the contribution of sialic acid and fructose in the formation of chromophore. The influence of furanose derivatives increases dramatically in hyperglycemic subjects, in whom the glycation of proteins can exceed normal values up to 4-fold (20,21). So, in diabetic patients it is quite necessary to examine the results of sialic acid colorimetric assays with certain care and consider the vast possibility of obtaining erroneously high levels. From this point of view, the thiobarbituric acid-based assay is most preferable, since molar extinction coefficient for fructose is negligibly low. On the opposite, Svennerholm's method (as well as other resorcinol-based assays) can hardly be used for LDL sialic acid determination in diabetics, since molar extinction coefficient for fructose is rather high, and the content of glycation products (generally called fructosamine) is comparable to that of sialic acid (21). Approximate calculations show that the LDL sialic acid estimates obtained by resorcinol assay may exceed real values even by 2.5-fold. Due to these considerations, we have abandoned the use of Svennerholm's method for LDL sialic acid determination in our laboratory.

The validity of correction introduced into standard Warren's assay was assessed in our recent work, where carbohydrate content of LDL was determined also by anion exchange chromatography using the pulsed amperometric detection method, and practically coinciding estimates have been obtained (16). Additionally, we have compared the data on LDL sialic acid content obtained in different studies (Table 2) (2,3,6,7,13,16,21-35). For better appearance, the values are expressed as mol sialic acid per mol LDL protein. It is known that apolipoprotein B has 20 potential N-glycosylation sites, of which 16 were found to be definitely glycosylated (36). Polysaccharide moiety of human apo-B consists of 5-6 mol high-mannose type and 8-10 mol complex (biantennary) oligosaccharides per mole apo-B protein (36). As biantennary type chain can bear two sialic acid residues, maximum theoretical value for protein-bound sialic acid level may account for 20 mol sialic acid per mol of apo-B protein. Additional quantities of sialic acid may be yielded from LDL lipid moiety. In our recent work we have shown that the level of lipid-bound sialic acid in LDL is 2-4 mol per mol of LDL protein (16). So, the maximum amount of total sialic acid in native LDL may be estimated as 24 moles per mol of LDL protein. The results of different studies shown in Table 2 are mainly in very good coincidence with the proposed theoretical value. In our early work (2), the reported results were moderately higher in comparison with other studies, but then the correction for sialic acid determination in LDL was not introduced yet, and the group of patients was rather small thus giving high deviation. The same considerations may be suggested for the results obtained by La Belle and Krauss (32). Incredibly high values of LDL sialic acid content were those of Melajarvi et al. obtained for healthy subjects and diabetic patients (35), that are in sharp dissonance with the data from other studies. The 3- to 15-fold excess of sialic acid as compared with usually observed levels may be explained only by evident inaccuracy of resorcinol-based determination method mainly used in that study. The evaluation of LDL sialic acid in diabetic patients group requires that any resorcinol-based assay should be avoided, since the principle of method itself does not allow to distinguish between sialic acid and furanosyl residues. Additionally, very long isolation procedure and the series of repeated ultracentrifugations could easily result in abundant accumulation of lipid peroxidation products in LDL that interfere in either method used, resorcinol-based or Warren's one. Such suggestion can easily explain the fact that sialic acid estimates in LDL subdivided into light, dense and very dense fractions were in any way significantly higher than in total LDL prior to separation by density gradient ultracentrifugation (35). The possibility of erroneous sialic acid determination by resorcinol-based assay was suspected of as far back as 1959, when Warren himself reported on the 1.4- to 5.4-fold difference in sialic acid estimates made by thiobarbituric acid and resorcinol methods (8). That time, the reason for such discrepancy was not explained, but it can be suggested that the sufficient amounts of products of non-enzymatic glycation were present in biological materials used for comparison (beef brain, beef liver, egg white etc.).

In conclusion, our experience in LDL sialic acid measurement allows to recommend modified thiobarbituric acid assay as relatively easy and non-expensive method. The correction procedure lets to avoid significant distortion of the results induced by the presence of interfering agents, mainly the products of lipid peroxidation. The more precise measurement of LDL sialic acid level would help to evaluate the diagnostic and prognostic significance of this parameter.

Aknowledgements

The research described in this publication was made possible in part by the Grant # 588 from Russian Council on Atherosclerosis and Grants ## 97-04-50124 and 97-04-48461 from Russian Foundation for Basic Research.

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Figure 1. Histogram showing the distribution of errors in LDL sialic acid determination by standard Warren's assay in LDL samples stored unfrozen for 1-5 days at 4°C (Panel A) and frozen for 1-8 weeks at -70°C prior to the assay (Panel B).

Figure 2. Graph showing the changes in resultant absorbance level determined by standard (Panel A, filled symbols) and modified (Panel B, hollow symbols) Warren assay. Sialic acid content was immediately measured in freshly isolated dialyzed LDL preparation obtained from normolipidemic healthy volunteer by two methods (standard and modified Warren's procedure). LDL aliquots of 50 ml containing 150 mg protein were stored at 4°C for 0, 1, 2, 3, 4 and 7 days and then frozen at -70°C to allow simultaneous measurement of sialic acid level. After 7 days, absorbance at 549 nm was determined under standard and modified Warren's procedure as described in "Materials and Methods" section. The data show the mean of three determinations ± S.E.M. Circles, LDL stored without antioxidant addition; squares, LDL stored in the presence of 1 mg/ml EDTA; diamonds, LDL stored in the presence of 0.1 mM butylated hydroxytoluene. Asterisk (*), the significant rise in absorbance as compared to initial level. Dashed lines, the 95% confidence interval for the initial absorbance level.

Figure 3. Graph showing the effect of LDL in vitro oxidation on resultant absorbance level determined by standard (filled circles) and modified (hollow circles) Warren assay. Sialic acid content was measured in freshly isolated LDL preparation obtained from normolipidemic healthy volunteer upon oxidation in the presence of 10^-5M Cu²⁺ at 37°C. The data show the mean of three determinations ± S.E.M. Asterisk (*), the significant rise in absorbance as compared to initial level. Dashed lines, the 95% confidence interval for the initial absorbance level.