Electrothermal Atomic Absorption Spectrometric Diagnosis of Familial

Anal. Chem. 2000, 72, 2406-2413

Electrothermal Atomic Absorption Spectrometric Diagnosis of Familial Hypercholesterolemia Jose´ Javier Martıń de Llano,*,† Enrique Jose´ Andreu,†,‡ Agustıń Pastor,§ Miguel de la Guardia,§ and Erwin Knecht†

Fundacio´ n Valenciana de Investigaciones Biome´ dicas, Instituto de Investigaciones Citolo´ gicas, Unidad de Medicina Molecular, Amadeo de Saboya 4, 46010 Valencia, Spain, and Department of Analytical Chemistry, University of Valencia, Dr. Moliner 50, 46100 Burjassot (Valencia), Spain

We have developed a new nonradioactive assay to identify human low-density lipoprotein receptor defects. It is based on the incubation of cultured cells with colloidal gold-LDL conjugates and quantitation of the gold associated with the cells by electrothermal atomic absorption spectrometry. After an oxidative treatment with nitric and hydrochloric acids, the biological matrix interferes neither with the gold recovery nor with the gold measurements, which are linear, at least from 0.15 to 3 ng of gold. When cells expressing a functional LDL receptor are incubated with increasing amounts of colloidal-gold LDL conjugates, the obtained saturation curve parallels that described when [125I]LDL is used as ligand. Moreover, this new assay allows us to clearly distinguish among fibroblasts from normal subjects or from heterozygous or homozygous patients of familial hypercholesterolemia, a very common autosomal disease. The assay is easy to perform, is sensitive, and avoids the use of radioactive compounds. Therefore, it could be successfully employed in the clinical diagnosis of this disease. Furthermore, since the methodology developed here can be applied to quantify the association of other gold-conjugated ligands to cells, it could have a widespread use in a variety of clinical and basic research studies. Familial hypercholesterolemia (FH) is a common autosomal dominant disease caused by defects in the low-density lipoprotein (LDL) receptor gene and with heterozygous and homozygous frequencies of about 1 in 500 and 1 in 1000 000 individuals, respectively.1 The mature receptor protein is located on the cell surface of different cells and binds and internalizes plasma LDL, a lipoprotein involved in cholesterol transport. After the internalization step in endosomes, the receptor releases the LDL particle, which is carried for degradation to lysosomes, while the receptor recycles back to the cell membrane where it can again bind a * Corresponding author: (e-mail) [email protected]; (fax) 34-96-360-1453. † Fundacion Valenciana de Investigaciones Biomedicas. ‡ Current address: Hospital Clıńico Universitario de Valencia, Avda. Vicente Blasco Ibań ˜ez 17, 46010 Valencia, Spain. § University of Valencia. (1) Goldstein, J. L.; Hobbs, H. H.; Brown, M. S. In The metabolic and molecular basis of inherited disease, 7th ed.; Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; Vol. II, pp 1981-2030.

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new LDL particle.2 A defective LDL receptor, as in FH, increases the plasma level of LDLs, which has been related to coronary heart disease.3,4 Clinically, FH is characterized by elevated LDL cholesterol levels in blood and by the presence of xanthomas, but some patients, especially children, do not show these alterations. Hence, when only considering these symptoms, it is not possible to reach an unequivocal diagnosis of the disease. Since an appropriate diet and drug treatment can reduce the risk of future coronary heart disease in FH patients, it would be advantageous to identify the affected subjects, particularly children, before the onset of the clinical symptoms. One approach to identify FH subjects is to find out, by genetic analysis, the nucleotide changes in the LDL receptor gene. However, this sometimes may require sequencing the whole gene. Besides that, a mutated LDL receptor gene does not necessarily imply that the receptor protein produced by the cell exhibits an altered function. Another diagnostic approach is the phenotypic study, based on determining the capacity to bind and internalize LDL particles of cells isolated from the subject, usually fibroblasts or lymphocytes. A dysfunctional LDL receptor, whichever the mutation is, will diminish these two processes. This kind of study, in addition to yielding information complementary to the genetic analysis, represents a faster, more accurate, and less expensive way to identify LDL receptor defects. Nevertheless, the phenotypic study has not found a broad diagnostic application in the clinical practice, mainly because in the classical assays the LDL particles were radioactively labeled with 125I.5 This isotope emits γ-radiation and requires appropriate equipment, a shielded area assigned for the use of radioisotopes, radioactive waste disposal facilities, and especially trained personnel for its handling. Several alternatives to 125I have been proposed. They include analysis by flow cytometry of fluorescent compounds, such as LDL labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI),6 antibodies conjugated to fluorescent dyes,7,8 (2) Brown, M. S.; Goldstein, J. L. Science 1986, 232, 34-47. (3) Hobbs, H. H.; Brown, M. S.; Goldstein, J. L. Hum. Mutat. 1992, 1, 445466. (4) Lestavel, S.; Fruchart, J. C. Cell. Mol. Biol. 1994, 40, 461-481. (5) Goldstein, J. L.; Basu, S. K.; Brown, M. S. Methods Enzymol. 1983, 98, 241260. (6) Verhoeye, F. R.; Descamps, O.; Husson, B.; Hondekijn, J.-C.; RonveauxDupal, M.-F.; Lontie, J. F.; Heller, F. R. J. Lipid Res. 1996, 37, 1377-1384. (7) Schmitz, G.; Bru ¨ ning, T.; Kovacs, E.; Barlage, S. Arterioscler. Thromb. 1993, 13, 1053-1065. 10.1021/ac991287p CCC: $19.00

© 2000 American Chemical Society Published on Web 04/25/2000

or analysis by other procedures of DiI-labeled LDL9 or europiumlabeled LDL.10 However, none of them has found a widespread use. We recently described a new approach based on the use of LDL conjugated to colloidal gold and inductively coupled plasma mass spectrometry (ICPMS) and discussed its advantages over other procedures.11,12 However, an inductively coupled plasma mass spectrometer, as originally employed, is a specialized and expensive piece of equipment which is not usually available in most hospitals and research departments. Moreover, since the feasibility of this assay was only tested with COS or CHO cells transfected with the human LDL receptor cDNA, it also remained to be investigated whether this procedure is a valid approach to study the expression of the LDL receptor in human cells. Therefore, in this paper, we describe an alternative nonradioactive method to measure the cellular gold content by electrothermal atomic absorption spectrometry (ETAAS). We also show that this assay is a valid procedure in the phenotypic diagnosis of FH, since it can distinguish between cell lines expressing or not a functional LDL receptor or among primary culture cells isolated from FH patients (both homozygous and heterozygous) and normal individuals. EXPERIMENTAL SECTION Gold Measurements. (a) Apparatus. The gold content of samples was determined using a Zeeman 4100 ZL atomic absorption spectrometer (Perkin-Elmer) with graphite furnace and a gold hollow-cathode lamp. The analytical wavelength used was 242.8 nm, and the slit was set to 0.7 nm. The Zeeman effect was used for background correction. New graphite tubes were conditioned by heating according to the manufacturer’s instructions. (b) General Procedure. Measurements were done by pipetting automatically a total volume of 30 µL on the L’vov platform of the graphite tube. The sequence in which the different solutions were taken up by the injector was as follows: matrix modifier (0.1% palladium and 0.06% magnesium nitrate in 1.5% HNO3, 5 µL), sample (up to 25 µL, always in multiples of 5 µL), and, if necessary, solvent (1.05% nitric acid, 1.2% hydrochloric acid, up to 25 µL). Two replicates were taken for each sample analyzed, except when specified. The furnace temperature program was optimized to obtain the highest and more consistent signal in the atomization step. The linearity of the measured integrated absorbances and the detection and quantitation limits were determined using serial dilutions of a commercial gold standard solution (1 g/L, Sigma) in an appropriate solvent. Standard solutions were kept in 5-mL polyethylene screw-cap tubes (Kartell) at 4 °C in the dark and used within 1 day. To oxidize both the biological material and the gold, a known volume of cellular lysate or of colloidal gold-LDL conjugate was (8) Raungaard, B.; Heath, F.; Brorholt-Petersen, J. U.; Jensen, H. K.; Faergeman, O. Clin. Chem. 1998, 44, 966-972. (9) Teupser, D.; Thiery, J.; Walli, A. K.; Seidel, D. Biochim. Biophys. Acta 1996, 1303, 193-198. (10) Wang, X.; Greilberger, J.; Ju ¨ rgens, G. Anal. Biochem. 1999, 267, 271278. (11) Martıń de Llano, J. J.; Andreu, E. J.; Knecht, E. Anal. Biochem. 1996, 243, 210-217. (12) Andreu, E. J.; Martıń de Llano, J. J.; Moreno, I.; Knecht, E. J. Histochem. Cytochem. 1998, 46, 1199-1201.

pipetted into 2-mL polypropylene microcentrifuge tubes (Costar) and brought to dryness using a model 5301 vacuum concentrator (Eppendorf). The day before the gold content was determined, the dried samples were treated with nitric and hydrochloric acids following the usual laboratory procedures for handling strong acids. In a fume hood, the tubes were uncapped and the acids were added. First, 30 µL of nitric acid (70%) was added, the tubes were capped and irradiated for 2 min at 80% of the maximal power in a 700-W household microwave oven. To avoid occasional uncapping while heating, the tubes were set in a plastic rack and the caps were kept pressed down using a laboratory label tape. After keeping the tubes for 2 min at room temperature, the heating and cooling steps were repeated twice. Tubes were allowed to cool to room temperature before they were centrifuged briefly in a microcentrifuge to recover any drops condensed on the tube walls. Then, 10 µL of hydrochloric acid (37%) was added and the tubes were capped and microwaved for 2 min as described above. After cooling, the tubes were briefly spun down and 1.96 mL of 1.036% hydrochloric acid was added to each one. Thus, final concentrations of nitric and hydrochloric acids were 1.05 and 1.2%, respectively. The oxidized samples were kept in the dark at 4 °C. To determine the influence of the cellular material on the recovery of the gold present in the samples, known amounts of a gold standard solution or of colloidal gold-LDL were added to tubes containing (or not) a pellet of 106 COS-7 cells. Samples were then dried and oxidized as described above. The possible interference of the oxidized cellular material in the measurements was also tested. Thus, 106 cells, with or without colloidal goldLDL, were dried, oxidized, and brought to 2 mL as described above. Then, 5-µL aliquots were automatically pipetted on the graphite tube together with 5, 10, or 15 µL of a gold standard solution (150 ng/mL), solvent (15, 10, or 5 µL, respectively) and 5 µL of the matrix modifier. Colloidal Gold-LDL Conjugates. Colloidal gold solutions were prepared from tetrachloroauric acid by established procedures.13 The mean diameter of the gold particles was determined on electron micrographs and using a grating replica. LDL particles were purified from serum of normolipidemic volunteers by sequential ultracentrifugation.5 To determine the nonspecific binding to cells (see below), LDL was extensively dialyzed against 150 mM NaCl/0.24 mM EDTA, pH 7.4, filter sterilized (0.45-µm Millex-HA filter, Millipore), and used within 1 week. For conjugation with colloidal gold, LDL was extensively dialyzed against 0.05% EDTA, pH 7.4, and used within 3 days. When required, LDL was stored frozen at -20 °C in the presence of 20% sucrose, as described.14 Protein content was calculated by a Lowry deoxycholate procedure.15 To conjugate LDL particles with colloidal gold, 5 mL of the gold suspension was added to 225 µL of LDL (1 mg/mL) in a glass tube, while briefly vortexing, and the suspension was incubated for 5 min at room temperature. To stabilize the gold colloid, 580 µL of 10% BSA (BSA fraction V), dissolved in double (13) Beesley, J. E. Colloidal gold: a new perspective for cytochemical marking; Royal Microscopical Society Handbook 17; Oxford University Press: Oxford, U. K., 1989. (14) Rumsey, S. C.; Galeano, N. F.; Arad, Y.; Deckelbaum, R. J. J. Lipid Res. 1992, 33, 1551-1561. (15) Harris, D. A. In Spectrophotometry and spectrofluorimetry: a practical approach; Harris, D. A., Bashford, C. L., Eds.; IRL Press: Oxford, U. K., 1987; pp 49-90.

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distilled water and sterile filtered, was added and mixed by inverting the tube several times. After 5 min at room temperature, the conjugates were put on ice. Several preparations were mixed, laid on a cushion of 20% sucrose in 150 mM NaCl/0.24 mM EDTA, pH 7.4, and ultracentrifuged at 31 500 rpm in a 60 Ti rotor (Beckman) for 1 h at 4 °C. The supernatant was discarded, and the reddish pellet was resuspended in the appropriate medium. For the binding and internalization assays, all colloidal gold solutions were used within 2 days. We have observed that colloidal gold-LDL conjugates, if frozen at -20 °C in the presence of 20% sucrose as described above for LDL, keep their functional properties for more than 1 year. When required, an aliquot of the frozen conjugate suspension was thawed and centrifuged and the pellet was resuspended in the appropriate medium. We routinely use these resuspended conjugates the same day they are thawed. For the binding and internalization assays, the gold conjugates were resuspended the same day in culture medium A (see below). Absorbance reading at 520-nm wavelength was used to roughly estimate the colloidal gold-LDL concentration. An aliquot of the colloidal gold-LDL conjugate was diluted (final absorbance at 520nm wavelength, 0.2-0.5) and the absorbance spectrum was recorded from 800 to 400 nm with a Helios Beta spectrometer (Unicam). To avoid the absorbance produced by phenol red (a pH indicator), the culture medium was used without this compound. The absorbance at 520 nm of the colloidal gold-LDL solutions employed in the assays was 0.75, 0.5, 0.25, or 0.0625. These solutions were prepared from the more concentrated preparations. In all the solutions, the actual gold content was exactly determined by ETAAS as described above. Mammalian Cell Culture. COS-7 cells, used to check for a possible interference of the biological matrix in the gold determination, were cultured as described.11 Cells were detached from the culture dishes using trypsin/EDTA solution (Life Technologies) and resuspended in phosphate-buffered saline (PBS). Cell density was estimated by counting in a hemocytometer. Aliquots of the cell suspension were centrifuged for 10 min at 100g in 2-mL tubes, the supernatants were discarded, and the cell pellets were dried. When required, a volume of gold standard solution or of colloidal gold-LDL was added to the cell pellet prior to the drying step. CHO-hLDLR, a cell line that expresses a functional human LDL receptor, and CHO-MOCK, a cell line that does not express a functional LDL receptor, were cultured as described.11,12 Cells were grown in Ham’s F12 media containing penicillin (100 units/mL), streptomycin (100 µg/mL), and 5% of fetal calf serum (FCS) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Lipoprotein-deficient FCS (LPDS), prepared as described,5 was used instead of FCS in the assay media (see below). Human fibroblasts were obtained from Coriell Cell Repository (Camden, NJ). The fibroblasts originated from a normal subject (GM003349B) and from FH patients, both heterozygous (GM00283 and GM01354) and homozygous (GM00488C). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, in MEM media containing penicillin and streptomycin, 10% FCS, and a 2-fold supplement of both essential and nonessential amino acids and vitamins. Colloidal Gold-LDL Binding and Internalization Assays. CHO-hLDLR and CHO-MOCK cells were seeded on 35-mm tissue 2408


culture dishes at a density of 30 000 cells/dish, and 2 mL of culture medium (Ham’s F12 containing penicillin and streptomycin and 5% of FCS) was added per dish. After 48 h, the medium was removed, 2 mL of fresh culture medium was added, and the cells were incubated for 48 h. Then, the medium was removed and it was replaced with 2 mL of Ham’s F12 medium containing antibiotics and 5% LPDS (culture medium A). Forty-eight hours later, the colloidal gold-LDL binding and internalization assays were done as follows. The medium was removed and it was replaced with 685 µL of fresh culture medium A. After a 20-min preincubation step at 37 °C and to determine the total binding and internalization of LDL, the following solutions were added to each dish, for a final volume of 1 mL: 65 µL of 150 mM NaCl/ 0.24 mM EDTA, pH 7.4, and 250 µL of the appropriate colloidal gold-LDL suspension. To determinate nonspecific binding and internalization, 65 µL of LDL (0.5 mg/mL, final concentration) was also added to some dishes just before adding the colloidal gold-LDL suspension. Blank dishes contained 65 µL of 150 mM NaCl/0.24 mM EDTA, pH 7.4, and 250 µL of culture medium A. All dishes were cultured for 3 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. After a 3-h incubation period, the cell dishes were put on ice, the medium was removed, and the cell monolayers were washed twice with ice-cold 50 mM Tris-HCl/150 mM NaCl, pH 7.4 buffer (TBS) containing 2 mg/mL BSA and twice with ice-cold TBS without BSA. Then, 400 µL of 0.5 mM EDTA, pH 7.4, was added to each dish and the dishes were incubated at 37 °C for 15 min. The swollen cells and the cell debris produced because of cell lysis were easily detached by pipetting the solution up and down several times. The lysate was transferred to a 2-mL microcentrifuge tube, 300 µL of fresh 0.5 mM EDTA, pH 7.4, was added to the dish, and after a 10-min incubation at 37 °C, this second volume was also incorporated into the microcentrifuge tube. The tube was vigorously vortexed and a 100-µL aliquot was transferred to a new tube which was used to measure the cellular protein. To quantify the gold content, another aliquot (250-500 µL) was transferred to a 2-mL microcentrifuge tube, dried, and processed as described above. For the colloidal gold-LDL binding and internalization assays with human fibroblasts, we followed the procedure described with [125I]LDL.5 Briefly, cells monolayers were trypsinized and the cells were seeded (day 0) on six-well plates (9.5 cm2 growth area, 30 000 cells, 2 mL of culture media/well). The medium was changed on days 3 and 5. On day 5, the new medium contained 10% LPDS instead of FCS. The binding and internalization assays were done on day 7 as described above for CHO cells, except that MEM was used instead of Ham’s F12 medium. Data analyses were performed using GraphPad Prism version 2.01 (GraphPad Software, San Diego CA). RESULTS AND DISCUSSION Colloidal gold particles conjugated with different ligands and antibodies by noncovalent bonds have been widely used in electron microscopy (EM) studies because of the electron-dense properties of gold.16 We and others have shown that in mammalian cells LDL particles conjugated with colloidal gold are bound to (16) Handley, D. A. In Colloidal gold. Principles, methods, and applications; Hayat, M. A., Ed.; Academic Press: San Diego, CA, 1989; pp 1-12.

Figure 1. Pyrolysis and atomization conditions for the determination of gold by ETAAS. Always, 5 µL of modifier, 5 µL of a gold standard solution (150 µg L-1), and 20 µL of a HCl and HNO3 blank solution were injected in succession. The atomization temperature for the pyrolysis studies was 2200 °C, and the pyrolysis temperature for the atomization studies was 1200 °C. The mean absorbance (b) and A-s value (9) obtained in duplicate determinations and the standard deviation, when not hidden by the symbols, are shown. Table 1. Temperature Program Used for the Measurement of Gold Content in Cultured Cells by ETAASa

dry dry pyrolysis atomization cleanup

temp (°C)

ramp (s)

constant (s)

110 130 1200 2200 2400

1 5 10 0 1

20 30 20 5 2

a The temperature at which the sample was loaded on the graphite furnace was 20 °C, and the absorbance measurements were carried out during the atomization step. A constant argon flow of 250 mL min-1 was employed, except during atomization, when argon flow was stopped.

and internalized by the LDL receptor.11,17 Furthermore, we have shown that ICPMS can accurately quantify the gold associated with cells expressing a functional LDL receptor. Since these measurements compared well with EM calculations,12 we concluded that this is a valid procedure to follow the binding and internalization of colloidal gold-LDL particles by cells. Here, we have tried to further improve this procedure and we have also investigated its feasibility in the routinary phenotypic diagnosis of FH. Measurements of Gold by ETAAS. First we tried to assess the suitability of ETAAS to measure gold in samples of cultured cells. As shown in Figure 1, pyrolysis and atomization temperatures of 1200 and 2200 °C, respectively, provided the highest absorbance values compatible with reduced thermal degradation of the graphite tubes. The furnace temperature program shown in Table 1 allowed a repetitive and consistent analysis of the gold samples at 242.8-nm wavelength. Typically, a graphite furnace was used for up to 200-250 measurements. After that, the size and shape of the absorbance peaks were considerably modified. Therefore, to check for a possible decrease in the performance of the graphite furnace, gold standard samples were always applied (17) Handley, D. A.; Arbeeny, C. M.; Witte, L. D.; Chien, S. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 368-371.

both at the beginning and at the end of every daily run. From measurements of blank samples (the integrated absorbance obtained from 10 different measurements was 0.0000 ( 0.0005, mean ( SD), the detection and the quantitation limits were calculated to be 13 and 44 pg, respectively (Table 2). The slope of the calibration curve (0.15-3 ng of gold, total volume 25 µL), obtained on six different days and using a new fresh preparation of the gold standard solutions each day, was 0.112 ( 0.003 ng-1 (mean ( SD; integrated absorbance increment/amount of gold increment). The gold content of samples with lower amounts of gold could be accurately calculated by drying the sample and dissolving it into a smaller volume or by directly drying several aliquots of the sample on the graphite furnace before the atomization step. Obviously, samples with more than 200 ppb gold can be diluted. Although the dynamic range for the quantitation of the gold content is narrower when ETAAS is used instead of ICPMS,11 the amplitude of the linear response interval obtained allows simultaneous analysis, in the same run, of samples with quite different gold contents. Moreover, and considering that in the cellular assay here described the sample is small, substitution of ETAAS for ICPMS has, among others, the main advantage of an increased sensitivity (10 times at least). This is because the concentration of the gold present in the ETAAS samples can be increased by drying, oxidizing, and dissolving them into a smaller volume (100-200 µL), while for the ICPMS measurement at least 1.5 mL of sample is consumed. To obtain consistent results, the presence of hydrochloric acid in the samples was necessary. This acid probably avoids the adsorption of gold on the tube surface. We have checked and found that there is no significant loss of gold from samples kept in plastic tubes for 25 days at 4 °C in the dark and in the presence of 1.05% nitric acid and 1.2% hydrochloric acid. Thus, the gold measured in standard solutions containing 100 and 200 ppb gold kept in 5-mL polyethylene tubes was 102 and 98% of the original values, respectively. Influence of Cells in the Gold Measurements. In these assays, COS-7 cells were used because we had previously observed that this cell line is more resistant to oxidation than other cell lines (e.g., CHO cells or human fibroblasts). We found that sequential incubation of 106 COS-7 cells pellets with small volumes of nitric and of hydrochloric acids, followed by a microwave treatment, as described in the Experimental Section, was a very effective procedure for the oxidation of the cellular material, since following treatment neither a pellet nor particulate material were observed. We noticed that it was important to add the acids separately, because if the nitric and hydrochloric acids were added together, the oxidation of the cellular sample was only partial. This oxidation step accomplishes the following: (i) hydrolysis of the cellular components, thus yielding a solution without particulate material, which reduces possible interferences of the biological matrix during the spectrometric detection of gold, and (ii) dissolving of the atomic gold present in the colloidal gold particles, consisting in a nucleus of crystalline gold.16 Although the oxidative treatment turn yellowish the white polypropylene tubes due to the absorption of nitrogen oxides, we never have observed a decrease in the integrity of the tubes that could affect the following processing steps. This oxidation treatment has been carried out in our laboratory for a period of more than 14 months without Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

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Table 2. Influence of Cells on the ETAAS Determination of Au calibration

Au content range (ng)

slope ( SD (ng-1)

Y-intercept ( SD

r2

Sy.x

detection limit (pg)

external in the presence of cells standard addition

0.044-2.25 0.047-2.25 0.95-3.20

0.123 ( 0.003 0.123 ( 0.003 0.118 ( 0.002

0.002 ( 0.004 0.007 ( 0.005 0.113 ( 0.002

0.999 0.998 0.999

0.005 0.006 0.003

13 14

observing acid leaks or any other problem. However, since this is not an approved standard procedure, the oxidation step could be completed more properly using special ovens and Teflon reactors.18 For blank samples derived from 106 oxidized COS-7 cells, the calculated detection and quantitation limits were 14 and 47 pg, respectively (Table 2). These values are similar to those obtained for blank samples without cells. As was the case with nonbiological samples (see above), there was no significant loss of gold content when the oxidized samples were kept at 4 °C in the dark. Thus, when an oxidized sample of cells incubated with colloidal goldLDL (equivalent gold content, 60 ppb) was kept for 25 days in a 2-mL polypropylene microcentrifuge tube, a 98% recovery was obtained. To determine whether the cells influence the gold recovery, aliquots of gold standard solutions or of colloidal gold-LDL were dried, with or without 106 cells, and oxidized before the gold content was determined. Thus, recoveries were 96 and 103% for samples containing cells and 50 and 300 ng of gold, respectively. If the oxidation step is omitted (i.e., the gold standard solution aliquot is dried and it is directly redissolved in 1.05% HNO3 and 1.2% HCl), the gold recovery is lower, ranging from 96 to 63% for samples containing 300 or 50 ng of gold, respectively. The oxidation step is even more important when samples containing colloidal gold-LDL conjugates are analyzed, which mimic the type of sample that will be processed after the assay to measure the association of colloidal gold-LDL with the cells. Thus, the percentage of gold recovered from a sample containing 350 ng of gold conjugates that was dried and redissolved in 1.05% HNO3 and 1.2% HCl was only 26% as compared with a sample that was oxidized after the drying step. Similarly to the results obtained with the gold standard solutions, when the same colloidal goldLDL sample was dried in the presence of 106 cells, 101% of the gold was recovered after the oxidation step. Another approach that was used to check for a possible influence of the oxidized cells in the gold measurements was to add, after oxidation, the same volume of cells, with or without colloidal gold-LDL, to various gold standard solutions. As shown in Table 2, the influence of the oxidized cells on the gold measurements is negligible. Thus, the slopes calculated for the lines obtained from the gold standard points, without or with a volume of hydrolyzed cells, are the same: 0.123 ( 0.003 ng-1. Likewise, when in the same measurement session a volume of a hydrolyzed sample of cells plus colloidal gold-LDL conjugates is added, the slope of the adjusted line is 0.118 ( 0.002 ng-1. When these regression lines were statistically compared, the slope values were not found to be significantly different. Thus, the method described herein allows us to determine the gold content of biological samples without any interference produced by the cellular material. (18) Begerow, J.; Dunemann, L. J. Anal. At. Spectrom. 1996, 11, 303-306.

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Quantitation of the Binding and Internalization of Colloidal Gold-LDL Conjugates by CHO Cells. In preliminary studies, we used colloidal gold-LDL conjugates in which the diameter of the gold particle was 17.1 ( 1.8 nm. We next tested in similar experiments several other gold sizes. We chose for the following experiments gold particles of 10.4 ( 0.6 nm diameter because this size increases the amount of the effective ligand (colloidal gold conjugated to LDL) keeping low the total amount of gold. Thus, about 3-5 LDL particles bind around each of these gold particles, instead of the 8-9 or 9-12 LDL particles observed, for example, surrounding 17-11 or 42-nm colloidal gold particles, respectively (Figure 2). Furthermore, we have observed that the nonspecific association of the 10.4-nm colloidal gold-LDL conjugates to cultured cells is lower than when colloidal gold of higher diameters was used. This could probably be due to the fact that an increase in the size of the gold particles also increases the surface area of the particle not covered by LDL particles, as determined by counting, for each gold size, the mean number of conjugated LDL particles per gold surface area. We have previously shown, measuring the gold content by ICPMS, that the gold associated with cells incubated with colloidal gold-LDL conjugates depends on the amount of receptor expressed by the cells11 and that the measured amount of gold truly accounts for the gold bound and internalized by the cells.12 To determine whether the high-affinity binding and internalization of colloidal gold-LDL conjugates to the cells can be described, as expected, by a first-order hyperbola, we carried out binding and internalization assays similar to those originally described by the group of Goldstein and Brown using [125I]LDL as ligand.5 After a 3-h incubation, the unbound colloidal gold-LDL was removed by several washes from the cell monolayers. The cell monolayers were detached from the dishes using 0.25 mM EDTA to recover all the cellular material and in order to keep to a minimum in the cellular samples the presence of salts, which could interfere in the ETAAS gold measurements. After a total incubation time of 25 min, the cells were completely detached and, most of them, lysed. The efficiency of this method was compared to others methods (e.g., employing a rubber policeman to mechanically detach the cell monolayers). The cell recovery, measured by protein content, was maximal using EDTA and equaled that obtained when NaOH was used to solubilize the cellular material (data not shown). Moreover, the use of EDTA does not interfere with the subsequent treatment of the samples with nitric and hydrochloric acids. The amount of gold associated with the CHO-hLDLR cells (referred hereafter as total gold) increased in a hyperbolic fashion (Figure 3). To determine the amount of gold associated with the cells by nonspecific binding and internalization of colloidal goldLDL conjugates (i.e., which is not receptor-mediated), duplicate cell dishes were incubated in the presence of an excess of LDL. As expected, the association of colloidal gold-LDL conjugates to

Figure 2. Gold-LDL complexes obtained with 42.0 (A) and 10.4 (B) nm colloidal gold particles. Purified human LDL was conjugated with colloidal gold as described in the text and processed for electron microscopy. About 9-12 (A) or 3-5 (B) LDLs bind to each electron-dense gold particle. In most of the conjugates, some of the LDL particles are hidden by the gold colloid and the other LDL particles. Bar, 0.1 µm.

Figure 3. Binding and internalization of colloidal gold-LDL conjugates by CHO-hLDLR and CHO-MOCK cells. Cells were cultured and incubated with gold-LDL conjugates and gold content was determined as described in the Experimental Section. The total gold associated to CHO-hLDLR (b) and CHO-MOCK (O) cells and the gold nonspecifically associated to these cells (9 and 0, respectively) are shown. Each experimental point is the mean of two measurements done in different cell dishes, and the standard deviation, when not hidden by the symbols, is shown as bars. The gold associated with high affinity to CHO-hLDLR cells was calculated as described in the text. The dashed line represents the hyperbolic curve that fits, by nonlinear regression, to the high-affinity data, from which the Amax and Kbi are calculated (see text).

CHO-hLDLR is a specific process, because it is effectively impaired by the unlabeled LDL present in excess in the media (Figure 3). When the amount of nonspecific associated ligand is represented against the concentration of the ligand in the medium, the experimental points can be adjusted to a straight line, with r2 ) 0.995 (Figure 3). The amount of gold nonspecifically associated with the cells at any ligand concentration was always less than 10% of the total gold. These values are similar or even smaller than those described for the nonspecific binding of [125I]LDL to adherent cells.5 The gold that is specifically associated with the cells is obtained by subtracting the nonspecifically associated gold from the total amount of gold bound and taken up by the cells. These values

correspond to the high-affinity binding of LDL particles to the LDL receptor, as previously described.5 Since our assay determines together the binding and the uptake of colloidal gold-LDL, we call Amax and Kbi the corresponding parameters here calculated for Bmax (maximum ligand bound) and KD (binding affinity; concentration of ligand that results in half-maximal binding), respectively, just to indicate that they include both processes. From the curve obtained when the high-affinity values are adjusted by nonlinear regression to a first degree hyperbola, it can be calculated that Amax and Kbi are 23.9 ng/µg (amount of gold per microgram of cellular protein) and 18.8 µg/mL (amount of gold per milliliter of cell culture medium), respectively. When these assays were done in parallel with CHO-MOCK cells, which do not express a functional LDL receptor,11 the ligand associated with the cells with high affinity was ∼2% of that associated with cells that express a functional LDL receptor (Figure 3). This is in agreement with the value reported in the literature for the activity of the LDL receptor expressed by the mutant CHO cell line.19 To compare the Kbi calculated here, using as ligand colloidal gold-LDL, with that estimated by others, using as ligand [125I]LDL, it is necessary to transform the amount of gold mass into amount of LDL protein mass. This can be done considering, as in many other immunogold procedures, that a particle of colloidal gold-LDL conjugate actually corresponds to one effective ligand particle which, in this case, is recognized by the LDL receptor molecule. Thus, from the mean diameter of the gold particle (10.4 nm) and the gold specific mass (19.3 g/mL), it can be calculated that a gold particle (volume, 5.9 × 10-19 mL) weighs 1.14 × 10-17 g and that the mass of 1 mol of gold particles is 6.9 × 106 g. Finally, the molecular weight of apolipoprotein B, the protein component of the LDL particle, is 5.1 × 105 and hence the equivalent LDL protein mass for a given gold mass can be (19) Kozarsky, K. F.; Brush, H. A.; Krieger, M. J. Cell. Biol. 1986, 102, 15671575.


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Figure 4. Binding and internalization of colloidal gold-LDL conjugates using fibroblasts from normal (b) subjects and from FH patients, both heterozygous (GM01354, [; GM00283, 1) and homozygous (9). Cells were cultured and incubated with gold-LDL conjugates and the gold content (associated gold: gold bound to and internalized by the cells) was determined as described in the Experimental Section. The gold nonspecifically associated with normal fibroblasts (O) is also shown. Each experimental point is the mean of measurements done in two different cell dishes, and the standard deviation, when not hidden by the symbols, is shown as bars. The gold associated with high affinity to the fibroblasts was calculated as described in the text. The dashed lines represent the hyperbolic curve fitted by nonlinear regression to the high-affinity data, from which the Amax and Kbi can be calculated (see text).

estimated by multiplying this value by 0.075 (5.1 × 105/6.9 × 106). Consequently, the Kbi for CHO-hLDLR cells (18.8 µg of Au/mL) would be 1.4 µg of LDL protein/mL. Quantitation of the Binding and Internalization of Colloidal Gold-LDL Conjugates by Human Fibroblasts from Normal Subjects and FH Patients. We finally investigated whether the new procedure developed here could be useful in the phenotypic diagnosis of patients with FH. A procedure similar to that developed for CHO cells was now performed to study the association of colloidal gold-LDL with human fibroblasts. Several parameters (seeding cell density, cell culture protocol, etc.) were also optimized here and thus, for example, it was shown that the cell monolayer can be efficiently detached by using 0.25 mM EDTA. The results obtained, when analyzing the binding and internalization of the colloidal gold-LDL conjugates to human fibroblasts, are similar to those described by Goldstein, Brown, and colleagues using [125I]LDL.2,5,20 Thus, the amount of gold nonspecifically associated with fibroblasts from a normal subject, which always represents less than 10% of the total gold associated with those cells, can be adjusted to a straight line (Figure 4). The amount of gold associated with high affinity to the cells increased in a hyperbolic fashion (Figure 4, dashed lines). From the firstdegree hyperbola, which can be adjusted to the experimental points by nonlinear regression, the Amax (20.7 ng/µg) and Kbi (14.0 µg/mL) can be calculated for normal fibroblasts. The total gold associated with homozygous FH fibroblasts was very low and close to the amount of gold nonspecifically associated with normal fibroblasts. When the experimental values are adjusted to a hyperbola, the Amax value calculated for the homozygous fibroblasts is less than 8% of the normal fibroblast value. For the fibroblasts from heterozygous FH patients, the high-affinity (20) Hobbs, H. H.; Brown, M. S.; Goldstein, J. L.; Russell, D. W. J. Biol. Chem. 1986, 261, 13114-13120.

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calculated values for Amax and Kbi were 18.1 ng/µg and 21.1 µg/ mL (GM01354 cells) and 5.1 ng/µg and 14.2 µg/mL (GM00283 cells), respectively. Even though the parameters calculated in that way are the result of a complex process, which involves both binding and internalization of the labeled LDL, the values calculated for the heterozygous subjects indicate that fibroblasts GM01354 have a mutant receptor with a lower affinity for LDL (compare the Kbi values calculated for fibroblasts from GM01354 and a normal subject: 21.1 and 14.0 µg/mL, respectively). The Kbi values calculated for the fibroblasts from the heterozygous GM00283 and the normal subject are equivalent (14.2 and 14.0 µg/mL, respectively), while the maximum association capacity value (Amax) for GM00283 is 5.1 ng/µg, which could imply that these cells exhibit a lower number of normal receptors than the fibroblasts from a normal subject (Amax ) 20.7 ng/µg). By calculations similar to those carried out with CHO cells (see above), we obtained a Kbi for normal fibroblasts (14.0 µg Au/mL) of 1.0 µg of LDL protein/mL. It is necessary to keep in mind that Kbi is the colloidal goldLDL concentration (expressed as gold or protein concentration) that results in half-maximal binding and internalization of the ligand. Although in most of the studies using [125I]LDL the published value is the concentration of [125I]LDL that results in half-maximal binding of the ligand,5 other studies show the concentration of [125I]LDL that results in half-maximal binding and internalization of the ligand and in half-maximal degradation of it. The last two parameters are similar to the Kbi constant calculated in our study. Thus, with cultured human fibroblasts incubated for 5 h at 37 °C, the concentration of [125I]LDL that resulted in half-maximal binding and internalization of LDL was 6.2 µg/mL21 and in half-maximal degradation was 5.4,21 4.4,22 or 3.6 µg/mL.23 These values are close to the Kbi value calculated in the present study (1.0 µg/mL) using a different and novel procedure. In summary, the method developed here allows one to differentiate fibroblasts obtained from normal individuals from those obtained from homozygous and heterozygous FH subjects that express a mutant LDL receptor. Furthermore, the association constant calculated for normal fibroblasts is similar to that described using [125I]LDL, supporting again the validity of this new method. Both methods follow the same schedule to culture cells and require the processing of similar amounts of cells for each assay point. After the incubation with the labeled ligand, both methods also require quantitation of the protein content. Although the quantitation of the gold can take a slightly longer period of time than the quantitation of the radioactivity, the method described here, besides avoiding the use of radioisotopes, presents further advantages. Thus, the LDL labeling procedure is mild when compared with the oxidation damage produced in the protein and lipid components of the LDL particle when labeling with 125I. Furthermore, [125I]LDL is unstable and must be used within a few days after the labeling step, while colloidal goldLDL conjugates can be stored for more than 1 year without (21) Fourie, A. M.; Coetzee, G. A.; Gevers, W.; van der Westhuyzen, D. R. Biochemistry 1992, 31, 12754-12759. (22) Chappell, D. A.; Fry, G. L.; Waknitz, M. A.; Muhonen, L. E.; Pladet, M. W. J. Biol. Chem. 1993, 268, 25487-25493. (23) Rubinsztein, D. C.; Coetzee, G. A.; Marais, A. D.; Leitersdorf, E.; Seftel, H. C.; van der Westhuyzen, D. R. J. Lipid Res. 1992, 33, 1647-1655.

noticeable changes in the binding properties to the LDL receptor. This allows preparation of large batches of labeled LDL to be used for a long period of time. Finally, the samples can be stored for prolonged periods, thus allowing one to simultaneously analyze a large number of samples obtained on different days or even months. Therefore, colloidal gold-LDL conjugates, as employed here, can be a safer and more convenient alternative to [125I]LDL for study of the binding of LDL to cultured cells. CONCLUSION A new procedure has been developed that allows the quantitation by ETAAS of colloidal gold present in biological samples. It has been applied to study the association of colloidal gold-labeled LDL to culture cells, and the results obtained with both CHO cell lines and human fibroblasts from normal and FH patients agree with those described by others using 125I-labeled LDL. The procedure is, at least, as sensitive and quick as the radioactive assay and avoids the use of radioisotopes. Since colloidal gold can be conjugated to a large variety of molecules,

this procedure, besides its applicability in the phenotypic diagnosis of FH, as shown here, can be easily adapted to many other important problems in clinical diagnosis and basic research. ACKNOWLEDGMENT We thank Isabel Moreno for her help in some of the previous experiments, El Himri Mamoune for his helpful advice on the ETAAS technique, and Asuncioń Montaner for technical assistance. This work was supported in part by Fondo de Investigacioń Sanitaria (FIS 96/2063 and 99/0008), Conselleria de Cultura Educacioń y Ciencia de la Generalitat Valenciana (GV97VS-23-111), Direccioń General de Ensen ãnza Superior e Investigacioń Cientı´fica (PB97-1445), and Fundacioń “La Caixa” (97/131).

Received for review November 10, 1999. Accepted March 6, 2000. AC991287P


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Electrothermal Atomic Absorption Spectrometric Diagnosis of Familial

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