NOËL ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 6, 2003 1225 RESIDUES AND TRACE ELEMENTS
Optimized Simultaneous Determination of Several Elements in Human Intestinal Caco-2 TC7 Cells by Inductively Coupled Plasma-Mass Spectrometry after Closed Vessel Microwave Digestion LAURENT NOËL, THIERRY GUÉRIN, and JEAN-MARC FRÉMY1 Agence Française de Sécurité Sanitaire des Aliments, Unité Contaminants de l’Environnement, Métaux Lourds et Eléments Minéraux, 10 Rue Pierre Curie, 94704, Maisons-Alfort, Paris, France HÉLÈNE HUET and MARTINE KOLF-CLAUW Laboratoire de Pharmacie-Toxicologie, Ecole Nationale Vétérinaire d’Alfort, 94704, Maisons-Alfort, Paris, France
A rapid procedure, based on closed vessels microwave digestion and inductively coupled plasma-mass spectrometry (ICP-MS), was evaluated to ascertain the effect of chronic exposure to cadmium on intracellular accumulation of minor and essential trace elements in cultured epithelial cells (Caco-2 TC7). For all measurements, the method of external calibration was used and 3 elements (Be, Sc, In) were selected as internal standards. Optimization procedures are discussed and results are presented for the total determination of 9 key analytes (Na, Mg, Ca, Cr, Mn, Cu, Zn, Mo, and Cd) in certified reference materials (CRMs) and 20 samples of Caco-2 TC7 cells long-term exposed to Cd. The performance characteristics of the analytical system were evaluated by calibration and linearity, limits of detection and quantitation, accuracy with spiking, trueness and repeatability with available CRMs. As a complement to the ICP-MS determinations, both available CRMs and cell samples were analyzed either by electro thermal- or flame atomic absorption spectrometry. The results were in good agreement with the ICP-MS results.
D
uring the last few years, increasing consideration has been given to interactions between the nutrition status of an organism and the degree of accumulation and toxicity of nonessential trace elements. These interactions are complex and involve biometals such as zinc (Zn), copper (Cu), chromium (Cr), magnesium (Mg), calcium (Ca), and some toxic elements, such as lead (Pb) or cadmium (Cd). For example, Cd is known to be one of the most toxic elements and is a major industrial and environmental pollutant. Its toxicity is based on its negative influence resulting from the Cd2+ Received April 4, 2003. Accepted by JS July 15, 2003. 1 Author to whom correspondence should be addressed; email:
[email protected].
substitution of other metal ions (mainly Zn2+, Cu2+, and Ca2+) in metalloenzymes and its very strong affinity to biological structures such as proteins, enzymes, and nucleic acids (1, 2). Cd enters the food chain (3), and concentrates within organisms due to its long biological half-life, 25–30 years in humans. This fact makes Cd a cumulative toxicant despite an intestinal absorption in humans estimated at about 5% (4). The intestinal epithelium is the main route of entrance for nutrients and, at the same time, the first barrier to be crossed, following oral administration of compounds such as food additives, drugs, and toxicants. The mechanism of metal absorption through these cells is of prime interest. In this context, human cell culture models are potentially useful to identify cellular mechanisms. The development of human cell culture systems has been limited by the loss of important in vivo anatomical and biochemical features. Attention has therefore turned to the use of human adenocarcinoma cell lines, such as HT-29 and Caco-2, that reproducibly display a number of characteristics of differentiated intestinal cells (5). Caco-2 cell line is currently one of the most widely used human cell culture models (6). Although established from a well-differentiated human colon adenocarcinoma cell line (7), Caco-2 cells exhibit spontaneous morphological and biochemical enterocytic differentiation at confluence in culture (8, 9). This differentiation process is growth-dependent and after confluency, brush border hydrolase activities are similar to those reported for normal villous enterocytes (10). The Caco-2 cell line, and particularly the late clone TC7, is a relevant in vitro model for studying human intestinal functions (6, 11), and has been widely used for the study of intestinal permeability and/or toxicity of several elements (Ca, Mg, Mn, Ni, Cu, Cd, and Zn; 12–16). Several methods and procedures are used for metal cell content determination in Caco-2 cells, including atomic absorption spectrometry (AAS), inductively coupled plasma-atomic emission spectrometry (ICP-AES), total reflection X-ray fluorescence spectrometry (TXRF), or multitracer radiolabeling for transport studies of selected elements. However, AAS is the most commonly used technique
1226 NOËL ET AL. : JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 6, 2003 Table 1. Instrumental parameters for ICP-MS Operating conditions Nebulizer
Apparatus
Concentric type pumped at 0.9 mL/min
Spray chamber
Scott-type double-pass water-cooled
Expansion stage
2.7 mbar
Intermediate stage
2.0 10–4 mbar
Analyzer stage
4.6 10–6 mbar
Sampling cone
Nickel, 1.0 mm orifice
Skimmer cone
Nickel, 0.75 mm orifice Standard mode (adjusted daily)
Plasma RF power
1350 W
Reflected power
