Workshop

Workshop Analytical Methods: Improvements, Advancements and New Horizons1 Carl L. Keen,* Thomas Jue,y Cuong D. Tran,** John Vogel,*z R. Gregory Downing,yy Venkatesh Iyengarzz and Robert B. Rucker*2 *Department of Nutrition, University of California, Davis, Davis, CA, yBiological Chemistry, School of Medicine, University of California, Davis, Davis, CA, **Gastroenterology Unit, Women’s and Children’s Hospital, North Adelaide, SA, Australia, zCenter for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, CA, yyR. G. D. Research, Inc., Niskayuna, New York and zzNutritional & Health-Related Environmental Studies Section International Atomic Energy Agency, Vienna, Austria ABSTRACT The workshop and exhibits dealing with analytical methods were selected to highlight the current state of the art in elemental analysis. The presentations in the first part of the workshop described approaches and advances important to the analysis of trace minerals. These presentations included: 1) two approaches to mass spectrometry, inductively coupled plasma and accelerator mass spectrometry; 2) use of nuclear magnetic resonance in studies of mineral function; and 3) the use and limitations of fluorescent probes in studies of metal uptake and regulation. In the second part of the workshop, the International Atomic Energy’s contributions to nutritional ‘‘metrology’’ were described. Advances in instrumentation over the past decade have led to extraordinary improvements in the precision and sensitivity of mineral analyses. The ability to address isotopic speciation at such low levels sets the stage for numerous novel approaches in the assessment of trace mineral function. J. Nutr. 133: 1574S–1578S, 2003. KEY WORDS:  fluorescent probes  nuclear magnetic resonance  inductively coupled plasma-mass spectrometry  accelerator mass spectrometry  metrology

ductively coupled plasma-mass spectrometry (ICP-MS)3. ICPMS is a versatile approach to mass spectrometry that provides high-quality multielements and isotopic analysis. The detection limit for most elements is in the subparts-per-billion range; some elements may even be detected in the parts-per-trillion range. Samples are introduced into a radio-frequency induced plasma. High temperatures at the center and the periphery of the plasma result in the conversion of most atoms into ions. The quadrupole configuration of commercially available ICPMS permits the detection of ions at each mass in rapid succession. Earlier methods that utilized neutron activation analysis or mass spectrometry of volatile metal chelates were generally not sufficiently accurate or precise. Thermal ionization mass spectrometry and ICP-MS are now the primary instruments used for isotope ratio measurements. Three excellent web sites are recommended that describe ICP-MS and its applications (1–3). Excellent reviews (4–6) may also be consulted for detailed descriptions. Accelerator mass spectrometry. Basic isotope labels can be sorted into two groups: 1) stable isotopes that occur at abundances of 0.1%–50% of the natural element, or 2) radioactive isotopes that occur at parts per billion or less. The sensitivity of isotopes as elemental or molecular tracers depends on the abundance, the background and the detection method. Stable isotopes are safe for laboratory use and human ingestion, but are limited in sensitivity by high natural abundances. Shortlived radioactive isotopes have high sensitivity in decay

Methodological approaches in the assessment of trace mineral analysis and function Inductively coupled plasma-mass spectrometry. Many of the exhibits associated with this workshop focused on in1 Presented as part of the 11th meeting of the international organization, ‘‘Trace Elements in Man and Animals (TEMA)’’ in Berkeley, California, June 2–6, 2002. This meeting was supported by grants from the National Institutes of Health and the U.S. Department of Agriculture, and by donations from Akzo Nobel Chemicals, Singapore; California Dried Plum Board, California; Cattlemen’s Beef Board and National Cattlemen’s Beef Association, Colorado; Clinical Nutrition Research Unit, University of California, Davis; Dairy Council of California, California; GlaxoSmithKline, New Jersey; International Atomic Energy Agency, Austria; International Copper Association, New York; International Life Sciences Institute Research Foundation, Washington, D.C.; International Zinc Association, Belgium; Mead Johnson Nutritionals, Indiana; Minute Maid Company, Texas; Perrier Vittel Water Institute, France; U.S. Borax Inc., California; USDA/ARS Western Human Nutrition Research Center, California; Wyeth-Ayerst Global Pharmaceuticals, Pennsylvania. Guest editors for the supplement publication were Janet C. King, USDA/ARS WHNRC and the University of California at Davis; Lindsay H. Allen, University of California at Davis; James R. Coughlin, Coughlin & Associates, Newport Coast, California; K. Michael Hambidge, University of Colorado, Denver; Carl L. Keen, University of California at Davis; Bo L. Lo¨nnerdal, University of California at Davis and Robert B. Rucker, University of California at Davis. 2 To whom correspondence should be addressed. Email: rbrucker@ucdavis. edu. 3 Abbreviations used: AMS, accelerator mass spectrometry; CRM, certified reference material; CRP, coordinated research project; IAEA, International Atomic Energy Agency; ICP-MS, inductively coupled plasma-mass spectrometry; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; m-PIXE, micro-proton-induced-X-ray emission; NIST, National Institute of Standards and Technology; NMR, nuclear magnetic resonance; RM, reference material; SRM, Standard Reference Materials.

0022-3166/03 $3.00 Ó 2003 American Society for Nutritional Sciences.

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counting above their very low backgrounds, but they can expose the experimental subjects to possibly harmful radiation. In contrast, although most long-lived isotopes have both low natural abundance and high sensitivity, they pose minimal radiation risks. Certain members of this last group are quantified by accelerator mass spectrometry (AMS), including 3H, 10 Be, 14C, 26Al, 32Si, 36Cl, 41Ca, 53Mn, 59, 63Ni, 60Fe, 99Tc, 129I and 242Pu. Decay counting is inefficient compared to mass spectrometric analyses of stable and very long-lived isotopes. One day of counting detects only 0.015% of 3H or 0.00034% of 14C present in a sample. Mass spectrometers quantify on the order of 1% of an isotope in a small sample. In this regard, AMS was developed and applied first to geochronology and archeology, only becoming a serious biochemical tool in 1990. AMS can quantify attomoles (10218 moles) or attocuries of elements or labeled compounds from milligram-sized biochemical samples. AMS brings three significant advantages to biochemical tracing: 1) high sensitivity for finding low-probability events or for use of subtoxic doses; 2) small sample size for painless biopsies or highly specific biochemical separation; and 3) reduction of overall radioisotope exposures, inventories and waste streams. AMS opens the door to increased chemical tracing in humans to obtain biochemical data about human health without uncertain extrapolations from animal models. Although light elements are quantified to attograms, heavier isotopes of metals and metalloids are detectable in 1–100 fg amounts. This sensitivity is a part in 104–106 of what is typically available in a 100-mL sample of blood or 100 mg tissue biopsy. An example of the power of AMS technology is its use in the estimation of the daily loss of calcium from the living skeleton. Administration of only nanocurie amounts of 41Ca, followed by a 6-mo delay, so that the fraction of the tracer not absorbed by the skeleton is cleared from the body, eventually leads to a steady state. The resorption of the labeled part of the skeleton can then be monitored by AMS detection of 41Ca resorbed and excreted for years after the tracer administration. Long-term evolution of the skeleton, as well as short-term perturbations, can then be easily studied. Micro-proton-induced-X-ray emission (m-PIXE) is another accelerator-based technology. m-PIXE quantifies elements (Z . Na) by counting elementally characteristic X-rays emitted from a defined spatial region when the sample is struck by a known amount of accelerated protons. m-PIXE analysis of thin film standards with biological samples quantifies elemental abundance to greater than 95% accuracy. The proton beam can be focused to under 1 mm in diameter for investigating detailed element distributions in single cells, isolated cellular components or tissue slices. A larger beam (50 mm diameter) may be used in scanning gels for metallo-proteins. m-PIXE directly quantifies metal ligands bound to proteins, and it can be useful in quantifying the amount of protein in the band, as well as determining some posttranslational modifications. The X-rays produced by sulfur and phosphorus concentrations are quantified above a well-characterized X-ray continuum at 1.9–2.1 keV and 2.2–2.4 keV, respectively, by m-PIXE. The sulfur content directly quantifies the concentration of cysteine and methionine, providing total quantification of proteins with known sequences. Phosphorylation fractions are derived directly from the phosphorus contents of a protein. The detection limit for elements commonly analyzed by m-PIXE is 1–10 ppm, where the total mass analyzed includes the substrate and the sample. Competing methods (atomic absorption spectroscopy, ICPMS (see above), or ion chromatography) for measuring metal contents of proteins require considerable preparation from

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milligram-sized fractions, whereas m-PIXE involves a simple deposition of sample on a support. Accelerator-based technologies provide sensitivity at parts per quadrillion for long-lived isotopes using AMS and at ppm concentrations for elemental abundances using m-PIXE [cf. references (7,8) for reviews]. Nuclear magnetic resonance (NMR). Magnetic resonance methodology presents a unique modality to capture tissue characteristics and metabolic processes without any invasive manipulation. Such an approach provides investigators with a means to study the impact of trace elements on the physiology and biochemical regulation of a variety of animal models and humans in vivo. Specifically, magnetic resonance imaging (MRI) can longitudinally track tissue morphometry as a function of experimental parameters, such as trace element levels, over an extended period in one model system. The images usually map the signal of water, which reflects the concentration and property of water in tissue, such that bone and tissue image contrast originates from the difference in water density and the vascular perfusion and tissue contrast arises from difference in water diffusion properties. Given the current engineering (high magnetic field strengths, magnet bore diameter, gradient strength, and the electronic instrumentation), researchers can follow volumetric tissue changes with micron pixel resolution. In fact, functional MRI techniques have interrogated cognitive function in response to different sensory stimulations. In addition to MRI, magnetic resonance spectroscopy (MRS) can monitor signals arising from cellular metabolites and lipids. Even though these signals have intensity ;100,000 times lower than water, they can be measured in vivo. Researchers have utilized the signals from 31P NMR to assess the bioenergetic status, from 1H NMR to monitor intracellular oxygenation and lipid status, and from 13C tracers to map a wide range of metabolic pathways in vivo, such as the glycolytic, pentose phosphate, glycogenolyic and fatty acid pathways. Unfortunately, trace element concentrations in tissue often fall well below the NMR detection limit. Thus, magnetic resonance techniques cannot directly detect trace element concentrations in vivo. However, MRI/MRS can monitor effectively the impact of trace elements on tissue growth and on metabolic regulation in a wide variety of models, spanning from cells, perfused organs, animals and humans. Given these perspectives, MRI/MRS can play a key role in understanding the impact of trace elements on cellular function (9–12). Fluorescent probes. Measurement of intracellular free zinc. The intracellular free zinc (Zn) is thought to be the pool of Zn utilized for biological function (13,14). However, consensus on the size of this pool varies widely, indicating that measurement of this entity is most likely beyond the capabilities of current technology. The potential for free Zn ions to form hydroxyl compounds and precipitate at physiological pH further adds to the enigma of the existence of free Zn ions. Reports of histochemical visualization and semiquantification of Zn in single cells and in tissues are now prominent, because of the development of Zn sensitive fluorimetric probes. Fura-2-based Ca21-sensitive fluorescent dyes have been used to measure intracellular Zn in cardiac myocytes (15), chromaffin cells (16) and in the nucleus and cytosol of bovine liver cells (17). However, these dyes have the limitation that they have a much greater sensitivity for calcium than for Zn, making specificity a significant issue. Zn-sensitive quinoline sulfonamide fluorescent probes (e.g., 6-methoxy-8 toluene sulfonamide quinoline) were developed in the early 1960s but they were not fully evaluated and made commercially available until the mid-1980s (18). However, solubility and the ability of quinoline sulfonamide probes to complex with Zn in mem-

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branes reduce their usefulness for semiquantitative measurements in living cells (19). More recently, two other quinoline derivatives have been developed; N- (6-methoxy-8quenolyl-carboxybenzoylsulfonamide); TFLZn, and methyl-8p-toluenesulphonamido-6-quinlyloxyacetic acid (Zinquin), which have much higher affinities for Zn. Zinquin is an ethyl ester derivative of quinoline, and it is relatively specific for Zn, with only cadmium causing weak fluorescence among many metals that were tested (20,21). Zn binds to Zinquin by complexing with either one or two nitrogen atoms to form complexes in a ratio of 1:1 (Zn: zinquin) or 1:2 (20,21). A unique property of Zinquin is that it is retained in living cells because the ethyl ester is cleaved by cytosolic esterase that imparts a negative charge that impedes its efflux across the plasma membrane. However, this may also impede its entry into the nucleus and cytoplasmic vesicles (20–22). Recently, Outten and O’Halloran (23) calculated from ligand exchange reactions that, in Escherichia coli, intracellular free Zn concentrations would be in femtomolar range (1016) with the caveat that they may not exist at all. Others have used various techniques to measure intracellular free Zn in mammalian cells with estimates that vary as much as six orders of magnitude. In erythrocytes, Simon (24), using 65Zn, estimated the intracellular free Zn pool to be 1.5–32 pM, whereas Adebodun and Post (25), using 19F NMR measurements in human leukemic cells, determined a concentration of 1 nM. In electrically stimulated chromaffin cells, free Zn concentrations monitored by fura-2 were estimated to be 0.4–2 nM (15). Brand and Kleineke (26) estimated that the free Zn pool in rat hepatocytes was in the range 0.6–2.7 mM, whereas in splenocytes and thymocytes, a value as high as 20–50 mM has been reported (20,21). Considering the wide range of free Zn concentrations that have been estimated with Zinquin, the question arises as to what is Zinquin measuring? Coyle et al. (22) showed in hepatocyte homogenates that Zinquin fluoresced with proteinbound Zn across a broad range of molecular weights. There was no evidence that Zinquin removed Zn from high molecular weight proteins In summary, the ability to measure free intracellular Zn pools may be more myth than reality, as this pool, if it exists, is too small to measure by present day technology. It seems most likely that Zn ions transverse the cytoplasm by ligand exchange mechanisms. Despite its limitation, Zinquin is a unique tool that provides high-resolution images of Zn in single cells and tissues. It is a rapid, inexpensive and highly reproducible method with high selectivity for Zn that can be used for visualizing and for the semiquantitative estimation of the Zn pool(s) that can be sequestered by the quinoline sulfonamide moiety. A new generation of Zinquin derivatives has been developed and is now being evaluated. It is hoped that these probes will permeate more of the intracellular compartments than Zinquin and be sufficiently sensitive to give more insight into the dynamics of the intracellular Zn pools. International Atomic Energy’s contributions to developments in nutritional metrology Metrology deals with the science of measurements that is devoted to the pursuit of identifying the most accurate results from an analytical effort. The expected outcome is a clear insight into the sources of errors quantified as uncertainties. Often the measurement process itself is benchmarked to a common reference point such as a certified reference material (CRM), a reference method or an SI unit (International System of Units) to safeguard the traceability aspect as exemplified by the document on metrology in chemistry and biology (27).

Several improvements have taken place in the past few decades contributing to measurable improvements in the analytical quality of results generated in many biomedical areas. The International Atomic Energy Agency (IAEA) has also greatly contributed over the past 30 years in a practical way to strengthen the analytical competence in the nutritional area, particularly in developing countries. The IAEA’s main emphasis has always been human capacity development as a basic factor, contributing to the thinking that a good analyst is the most important component of any analytical system. This requires that the analyst understands that a reliable analytical result is the product of a valid sample that is analyzed with due precautions. Ensuring that analytical results generated in field studies meet the desired expectations has been a great concern for the IAEA. In the course of implementing a wide variety of projects over the years, the IAEA has gone through a process of continuous refinement, modeled on accumulating practical experience. The process, mainly operating through the IAEA’s Coordinated Research Project (CRP) mechanism, is the result of a long and enduring learning curve for both the agency and the participants, as numerous difficulties unfolded along the way. A sequence of examples spread over the past three decades illustrates the positive developments that have taken place and improved the metrological profiles of the results generated by field studies supported by the IAEA. A study on trace elements in cardiovascular diseases initiated by the IAEA in the early 1970s did not completely achieve the set goals due to two reasons: noncompliance by the participants in adopting harmonized sampling protocols developed by the IAEA, and improper methods used for analysis. As a result, the IAEA enhanced its focus on analytical quality assurance as a whole in the 1980s resulting in the introduction of a range of diversematrix CRM (e.g., total diet material, milk powder, among others). After this, a project on determination of toxic trace elements in foods was initiated and some improvements were noticed in the overall situation. However, data evaluation of this project revealed unsatisfactory variance in results even though the laboratories had used similar analytical methods on comparable food samples indicating the need for further improvements. Subsequently, in the late 1980s sampling and sample preparation along with proficiency testing assumed high priority under IAEA activities, and projects involving intercomparison runs were undertaken. Substantial progress was also achieved by providing standardized equipment made of titanium knives and special blenders for minimizing contamination, under the ‘‘controlled contamination’’ approach. Simultaneously, another change in focus by recognizing the importance of matrix specific reference materials (RM) (human milk matrix versus cow milk matrix) helped to identify influence of sample matrix on the accuracy of analytical results. In addition, quite recently existing RM (e.g., NIST SRM bone meal, total diet and pine needles) were taken up for upgrading certification to meet project specific needs (e.g., generating reference values for Cs, I, Sr, Th and U) that were specifically identified for the project on Reference Asian Man (28). By the 1990s, all of these efforts collectively contributed to measurable improvements in the quality of analytical results. The ‘‘harmonization’’ of relevant parameters was the next step implemented under the IAEA field projects by pooling the experiences gained over a number of years that made the whole process of analytical quality assurance very robust (29). The parameters harmonized included design of the project protocol, sampling and sample processing procedures, streamlined

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TABLE 1 Selected sources of bias in human experiments involving stable isotope techniques Study part Study design Subjects Isotope administration

Sampling Sample preparation Mass spectrometry Data evaluation

Source of bias

Potential cause of bias

Choice of method Isotope doses Number Inclusion/exclusion criteria Isotopic labeling Oral dose Intravenous dose Subject control Time schedule Contamination Homogenization Isotope effects Multiple Choice of model Data transformation Data modeling Uncertainty estimate

Method might not be suitable to answer question Insufficient isotopic enrichment of sample material Effect is hidden by interindividual variations Interferences with measured parameter No isotopic equilibration between native element and isotopic label in the gut or body Incomplete intake of isotopic label Incomplete transfer; metabolism of oral and isotopic label differs No fasting before/after isotope administration; incomplete sample collection by the subject Sampling started too early, too short or was not long enough Over-/underestimation of isotopic label in sample Isotopic composition of sample is not representative Systematic changes in isotopic composition Multiple Unjustifiable assumptions/simplifications Unjustifiable assumptions/simplifications Technique by which data are fitted to metabolic model Not all sources of analytical and physiological relevance have been considered

approaches for validation of methods and data acquisition, and appropriate strategies for data treatment. These steps were linked to a so-called ‘‘reference laboratory’’ concept where the same validated method was used for determining a given analyte for all samples of a CRP. These steps were instrumental in generating data usable for global recommendations as witnessed by the six-country World Health Organization (WHO) human milk project (30) and the multicountry global dietary intake project providing baseline intake values applicable for global populations as reflected in the WHO/Food and Agricultural Organization (FAO)/IAEA report (31). In the late 1990s by introducing the concept of ‘‘central laboratory’’, the reliability level of field project results improved further. Under this concept, 10% of all samples generated within a project was centrally analyzed by an established laboratory in the region with access to multiple analytical methods, while the participants’ laboratories carried out the bulk of the work under good laboratory practice conditions. This effort has yielded excellent results even for difficult to determine trace elements such as Cs, I, Th and U (radiologically important ones, some at subparts per billion level), as witnessed in the recently completed CRP on Reference Asian Man (32, 33). Similar metrological improvements have also been recorded for measurements of importance in human physiology. Comparable measurements of bone mineral densities in subjects belonging to 11 countries using dual energy X-ray absorptiometry based on two measurement modes (lunar and hologic) have been documented in a recently completed CRP on osteoporosis (34). This is the first set of harmonized measurements on a large scale. Similarly, a currently running CRP on Ageing and Obesity has identified systematic errors of over 10% in dietary energy intake by applying the doubly labeled water technique (35). Importantly, results from these investigations have supported the FAO/WHO/United Nations University expert committee deliberations currently taking place to establish new energy recommendations including rural populations. The light isotope users group has dealt with standardization and harmonization issues for a long time and there is a growing feeling that a similar grouping of heavy isotope users would be very beneficial. Recognizing this the IAEA called for a workshop of the stable isotope users group during the International Congress of Nutrition in 2001. The current status of some

stable isotope methods used in nutrition research and several sources of bias in human experiments involving stable isotope techniques were identified (36). Tables 1 and 2 summarize the essence of the discussions that took place, which goes a long way in understanding the factors contributing to sources of errors in stable isotope measurements. To apply stable isotope techniques successfully, potential sources of error in the study design, the experimental part, sample analysis and data transformation have to be identified beforehand and, if possible, suitable measures be taken for bias control. Although sources of bias in the analytical part and in the calculations can be identified and eliminated before running the human experiment, sources of bias in the study design and the experimental part are often hidden or difficult to assess. Thus, successful

TABLE 2 Current status of some stable isotope methods used in nutrition research Method (2H, 18O)-techniques Energy expenditure Body composition (2H) Breast milk intake

Standardization status Good level of standardization No formal standards but these could easily be achieved No formal standards but these could easily be achieved

13

C Breath tests 13 C-urea (Helicobacter pylori) 13 C-octanoate (gastric emptying) Nutrient assimilation Gut transit times Liver function

Any gas chromatography/ Combustion/Isotope ratio mass spectrometry method Most gas chromatograpy/ Mass spectrometry methods Any mineral and trace element technique

Good level of standardization Some standardization, could be improved Some standardization, could be improved Some standardization, could be improved No significant standardization Uniform approaches but no standardization Uniform approaches but no standardization Uniform approaches but no standardization

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application of stable isotope techniques depends on the expertise of the team of scientists running the experiment. Additionally, development of a user friendly natural matrix RM database (http://www.iaea.or.at/programmes/nahunet/e4/ index.html) exclusively for environmental, biological and food matrix based RM, analytical workshops, projects contributing to training and fellowship programs, symposia, workshops and conferences are collectively responsible for the metrological improvements reflected in nutrition and environment projects as a result of persistent involvement of IAEA with developing countries as documented (37). LITERATURE CITED 1. http://www.chem.agilent.com/ 2. http://instruments.perkinelmer.com/ 3. http://www.micromass.co.uk/ 4. Vanhaecke, F., Resano, M. & Moens, L. (2002) Electrothermal vaporization ICP-mass spectrometry (ETV-ICP-MS) for the determination and speciation of trace elements in solid samples - A review of real-life applications from the author’s lab. Anal. Bioanal. Chem. 374: 188–195. 5. Sutton, K. L. & Caruso, J. A. (1999) Liquid chromatography-inductively coupled plasma mass spectrometry. J. Chromatogr. 856: 243–258. 6. Barnes, R. M. (1998) Plasma source mass spectrometry in experimental nutrition. Adv. Exp. Med. Biol. 445: 379–396. 7. Turteltaub, K. W. & Vogel, J. S. (2000) Bioanalytical applications of accelerator mass spectrometry for pharmaceutical research. Curr. Pharm. Des. 6: 991–1007. 8. Vogel, J. S. & Turteltaub, K. W. (1998) Accelerator mass spectrometry as a bioanalytical tool for nutritional research. Adv. Exp. Med. Biol. 445: 397–410. 9. Tran, T. K., Sailasuta, N., Kreutzer, U., Hurd, R., Chung, Y., Mole, P., Kuno, S. & Jue, T. (1999) Comparative analysis of NMR and NIRS measurements of intracellular PO2 in human skeletal muscle. Am. J. Physiol. 276: R1682– R1690. 10. Heerschap, A., Houtman, C., in ‘t Zandt, H. J., VAN den Bergh, A. J. & Wieringa, B. (1999) Introduction to in vivo 31P magnetic resonance spectroscopy of (human) skeletal muscle. Proc. Nutr. Soc. 58: 861–870. 11. Roden, M. & Shulman, G. I. (1999) Applications of NMR spectroscopy to study muscle glycogen metabolism in man. Annu. Rev. Med. 50: 277–290. 12. Kay, L. E. (1998) Protein dynamics from NMR. Biochem. Cell Biol. 76: 145–152. 13. Tran, C. D., Butler, R. N., Howarth, G. S., Philcox, J. C., Rofe, A. M. & Coyle, P. (1999) Regional distribution and localization of zinc and metallothionein in the intestine of rats fed diets differing in zinc content. Scand. J. Gastroenterol. 34: 689–695. 14. Tran, C. D., Butler, R. N., Philcox, J. C., Rofe, A. M., Howarth, G. S. & Coyle, P. (1995) Regional distribution of metallothionein and zinc in the mouse gut: comparison with metallothionein-null mice. Biol. Trace Elem. Res. 63: 239– 251. 15. Atar, D., Backx, P. H., Appel, M. M., Gao, W. D. & Marban, E. (1995) Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J. Biol. Chem. 270: 2473–2477. 16. Vega, M. T., Villalobos, C., Garrido, B., Gandia, L., Bulbena, O., GarciaSancho, J., Garcia, A. G. & Artalejo, A. R. (1994) Permeation by zinc of bovine chromaffin cell calcium channels: relevance to secretion. Pflugers Arch. 429: 231– 239. 17. Hechtenberg, S. & Beyersmann, D. (1993) Differential control of free calcium and free zinc levels in isolated bovine liver nuclei. Biochem. J. 289: 757– 760. 18. Frederickson, C. J., Kasarskis, E. J., Ringo, D. & Frederickson, R. E. (1987) A quinoline fluorescence method for visualizing and assaying the

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