Anal. Chem. 1998, 70, 2218-2220
Articles
Microwave Method for Preparing Erythrocytes for Measurement of Zinc Concentration and Zinc Stable Isotope Enrichment John W. Huffer,* Jamie E. Westcott, Leland V. Miller, and Nancy F. Krebs
Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado 80262
A microwave digestion method to prepare human erythrocytes for measurement of Zn concentration by atomic absorption spectrophotometry and stable isotope enrichment by mass spectrometry is described. Also described is a process for purifying digested erythrocyte samples enriched with Zn stable isotope for analysis by fast atom bombardment mass spectrometry. Microwave digestion was investigated as a way to increase sample throughput by replacing a more time-consuming conventional oven ashing/hot plate wet digestion method. Pooled red blood cells and NIST bovine liver standard reference material were digested by the two different methods and zinc recoveries compared by atomic absorption spectrophotometry. Microwave and conventional methods yielded 11.7 ( 0.1 and 11.7 ( 0.2 µg/g (wet wt), respectively, for the pooled erythrocytes, and Zn recovery from NIST bovine liver standard (certified 123 ( 8 µg/g) was 128.2 ( 1.2 and 127.4 ( 1.3 µg/g, p g 0.282, respectively. Microwave digestion improved the processing of erythrocytes for atomic absorption spectrophotometry and mass spectrometry by reducing digestion time from 1 week to 2 h. In addition, a procedure for purifying digested erythrocyte samples by ether extraction and ion-exchange chromatography in preparation for mass spectrometry analysis of Zn stable isotope enrichment is outlined. There is rapidly growing appreciation of the importance of zinc in human biology and of the practical implications of zinc deficiency for human growth and development.1 Adequate understanding of factors that affect zinc homeostasis and of how subtle changes in zinc nutritional status are linked to pathophysiological and clinical changes depends, in part, on a better understanding of whole body zinc metabolism. The application of zinc stable isotope techniques, especially combined with modelbased compartmental analysis, has opened up new avenues for advancing our understanding of human zinc metabolism under a variety of physiologic and pathologic circumstances. Erythrocytes are one of the few accessible tissues of humans for measuring (1) AJCN Supplement: Zn for Child Health: Symposium Proceedings, Baltimore, MD, Nov 17-19, 1996; Black, R. E., Kelley, L. M., Eds.; American Journal of Clinical Nutrition: Bethesda, MD, 1998 (in press).
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isotope enrichment. Moreover, erythrocytes have been reported to be a site of regulation for zinc metabolism.2 Processing of biological samples for mass spectrometry analysis of zinc stable isotope ratios typically requires digestion of the organic matrixes in which zinc is found, followed by isolation and purification of the metal from other elements present in the original tissue. We report a microwave digestion method for preparing red blood cells for measurement of zinc concentration and zinc stable isotope enrichment with the goal of improving sample throughput. EXPERIMENTAL SECTION Study Design: Comparison of Two Digestion Methods for Zinc Concentration Analysis. The ashing/hot plate method involves (i) drying the material in a drying oven, (ii) ashing at high temperatures, (iii) performing a wet digestion in nitric acid on a hot plate, (iv) ashing again at high temperature, and (v) reconstituting in hydrochloric acid. The microwave method involves (i) mixing the samples with concentrated nitric acid in advanced composite vessels, (ii) ramping the mixtures to a maximum temperature and pressure, (iii) transferring them to a glass beaker, (iv) evaporating them over a hot plate, and (v) reconstituting them in hydrochloric acid. A comparison of zinc recoveries, as determined by atomic absorption spectrophotometry, was carried out between the microwave method and the ashing method using aliquots taken from a pool of non-isotopically enriched red blood cells. Zinc recovery was also tested by each method using a National Institute of Standards and Technology standard reference material. Application of the Method. Red blood cells from human subjects given intravenous and oral doses enriched in 70Zn were digested by the microwave method, purified, and analyzed by fast atom bombardment mass spectrometry. Red Blood Cell Samples. A red blood cell pool was collected from seven healthy laboratory personnel who donated a total of 300 mL of whole blood. Individual samples were added to heparinized vials and subsequently centrifuged at 1600g for 10 min. The plasma and buffy layer were removed by aspiration with a plastic transfer pipet, and the red blood cells were washed 5× (2) Wastney, M. E.; Aamodt, R. L.; Rumble, W. F.; Henkin, R. I. Am. J. Physiol. 1986, 251 (Regulatory Integrative Comput. Physiol. 20), R398-R408. S0003-2700(97)01083-4 CCC: $15.00
© 1998 American Chemical Society Published on Web 05/05/1998
in normal saline solution. All samples were then pooled and suspended in triple-filtered deionized water (Milli-Q) in a ratio of 1:1 by weight. The red blood cell suspension was stored at -20 °C until further processing. Red blood cells enriched with the stable isotope 70Zn were obtained by venipuncture from five healthy adults enrolled in a study of zinc metabolism and kinetics. Serially drawn blood samples following intravenous 70Zn isotope administration were initially handled as described above, digested according to the microwave method, and then purified for fast atom bombardment mass spectrometry. Instrumentation. Total zinc recovery was measured using a Perkin-Elmer model 2380 atomic absorption spectrophotometer equipped with a deuterium arc background correction lamp and a Perkin-Elmer Ca-Mg-Zn Intensitron lamp (Perkin-Elmer Corp., Norwalk, CT). The linear calibration curve was set using 0.70 and 1.25 ppm zinc standards in 0.125 N hydrochloric acid. Measurement of red blood cell 70Zn enrichment was carried out using a VG 7070E HF double-focusing mass spectrometer (VG Analytical, Manchester, UK) equipped with an Ion Tech atom gun (London, UK). Microwave digestion was performed in an MDS-2000 (CEM Corp., Mathews, NC) microwave sample preparation system. The 12 Advanced Composite Vessels were capable of performing at a maximum operating temperature and pressure of 200 °C and 200 psi. One of the Advanced Composite Vessels was equipped with a modified lid, designed for use with pressure/temperature sensing equipment. Conventional oven ashing was accomplished in a model 51894 box furnace (Lindberg, Watertown, WI). All laboratory equipment used in sample preparation was acid washed and rinsed in deionized water, as outlined by Peirce et al.,3 in order to avoid contamination by naturally occurring zinc. All laboratory materials used in this study were free of detectable zinc as determined by atomic absorption spectrophotometry. Chemicals and Reagents. All water (Milli-Q) used in sample processing and reagent production was triple-filtered, deionized, and zinc-free (Millipore Continental Water Systems, Bedford, MA) as determined by atomic absorption spectrophotometry. Concentrated trace metal grade hydrochloric and nitric acid stocks were manufactured by Seastar Chemical Inc. (Pittsburgh, PA). Diisopropyl ether was manufactured by Fisher Chemical (Fairlawn, NJ). Ion-exchange resin AG 1-X8 resin 100-200 mesh chloride form was manufactured by Bio-Rad Laboratories (Hercules, CA). Chromatography columns (Bio-Rad) had a 10-mL capacity with a resin volume of roughly 1 mL. METHOD DEVELOPMENT Comparison of Zinc Recoveries. Digestion of red blood cells by the conventional ashing method was carried out by weighing 24 accurately measured aliquots (approximately 5 g) of red blood cell suspension into glass beakers. After desiccation in a drying oven at 80 °C for 24 h, the aliquots were ashed in a muffle-furnace at 200 °C for 4 h, 250 °C for 1 h, 300 °C for 20 h, 350 °C for 1 h, and 450 °C for 24 h. The gradual, controlled temperature increase prevented samples from overflowing their containers as a result (3) Peirce, P. L.; Hambidge, K. M.; Goss, C. H.; Miller, L. V.; Fennessey, P. V. Anal. Chem. 1987, 59, 2034.
Table 1. Time, Temperature, and Pressure for Microwave Digestion of Erythrocytes cycle 1
cycle 2
stage stage stage stage stage stage stage stage 1 2 3 4 1 2 3 4 power (W) pressure (psi) temperature (°C) time (min) ramp time (min)
25 50 100 3 15
25 75 123 3 15
50 100 130 3 15
100 125 140 3 15
100 85 150 3 15
100 140 165 3 15
100 185 185 3 15
100 185 185 3 15
of initial violent combustion. Wet digestion in concentrated nitric acid on a hot plate for 15 min preceded an additional 24 h of ashing at 450 °C. Ashed samples were reconstituted in 10 mL of 6 N hydrochloric acid. After dilution with Milli-Q water, samples were analyzed for zinc content by atomic absorption spectrophotometry. Microwave digestion was performed on cells taken from the same pool as those used for the ashing method. Twenty-four accurately weighed aliquots (approximately 5 g) of red blood cell suspension were placed into Advanced Composite Vessels, combined with 5 mL of concentrated nitric acid, and ramped to a maximum temperature and pressure of 185 °C and 185 psi, using the MDS-2000 (Table 1). Digested samples were transferred to glass beakers, evaporated to dryness on a hot plate, and reconstituted in 10 mL of 6 N hydrochloric acid. Dilutions of the red blood cells from the microwave method and the ashing method were measured and compared for zinc concentration by atomic absorption spectrophotometry. Validation of Recovery. Recovery of zinc using ashing and microwave digestion was compared using NIST Standard Reference Material Bovine Liver 1577a, with a certified zinc concentration of 123 ( 8 µg/g. Weighed aliquots of standard were processed by the microwave method and the ashing methods described above. Application. The microwave method was used for samples enriched with 70Zn isotope and the samples were analyzed for 70Zn enrichment by fast atom bombardment mass spectrometry. After the digestion and before the measurement of isotopic enrichment, steps were taken to remove iron and other inorganic elements from the samples. The purification steps included (i) transferring the digested samples into glass beakers, (ii) evaporating the nitric acid on a hot plate, (iii) reconstituting the samples in 8 N hydrochloric acid, (iv) extracting >97% of aqueous iron from the solution by mixing with 5 mL of diisopropyl ether (2×), (v) evaporating the solution to dryness to eliminate residual dissolved ether, (vi) reconstituting the sample in 6 N hydrochloric acid, and (vii) removing the remaining iron and other inorganic elements by ion-exchange chromatography (2×). The final ∼4 mL of zinc-containing eluents, collected in polyethylene test tubes, were measured by atomic absorption spectrophotometry to calculate approximate total zinc concentrations before evaporation in a vacuum centrifuge. The final zinc concentration calculations were used to reconstitute in enough 0.125 N hydrochloric acid to create highly purified, isotopically enriched samples within a zinc concentration range of 60-100 ppm. The samples were then measured for enrichment of the stable isotope 70Zn by fast atom bombardment mass spectrometry. Analytical Chemistry, Vol. 70, No. 11, June 1, 1998
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RESULTS AND DISCUSSION Method Development: Comparison of Zinc Recoveries. Forty-eight samples from the red blood cell pool were processed, 24 by the microwave method, and 24 by the ashing method, with zinc recoveries essentially identical at 11.7 ( 0.1 and 11.7 ( 0.2 µg/g (mean ( SD) for microwave and conventional methods, respectively. Ten NIST bovine liver samples were processed by the microwave method and 10 by the ashing method, with recoveries of 128.2 ( 1.2 and 127.4 ( 1.3 µg/g, respectively, p g 0.282. Throughput. Although the ashing oven can process more samples at one time than the microwave digestor, throughput can be up to 5 times higher using microwave digestion. Microwave digestion requires less than 8 h per 48 samples versus 4-5 days per ∼50 samples for the ashing method (amount of time needed to digest or ash only). As accurately as the atomic absorption spectrophotometry limits of detection can discriminate, advanced composite vessels may be used repeatedly to process multiple sample sets within a short time, with no cross contamination from previous samples to subsequent samples. However, more sensitive studies using isotope ratio measurements will be needed to assess whether samples containing very low levels of isotopic enrichment may be vulnerable to “relative isotopic abundance contamination” from previous samples which are highly enriched in one or more isotopes. Extraction and Cleanup. The cleanup procedure helped eliminate interference caused by the presence of elements other than zinc in measuring isotope peak ratios on the mass spectrometer. Red blood cell samples contain abundant hemoglobin iron, whose atoms and oxides pose a significant source of zinc isotope signal interference in fast atom bombardment mass spectrometry. Published and unpublished data indicate that optimum iron extraction occurs in ∼8 N hydrochloric acid.4 Two extractions removed the majority (97-99%) of iron, but subsequent extractions removed too little of the remaining iron to justify a third extraction. After the extraction step, samples were evaporated to dryness again to eliminate any remaining dissolved ether. The remaining (4) Dodson, R. W.; Forney, G. J.; Swift, E. J. Chem. Soc. 1936, 175, 25732577.
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iron and other competitively interfering metals such as sodium, calcium, and potassium were removed by ion-exchange chromatography. Samples passed through ion-exchange columns only one time produced poor peaks when measured by fast atom bombardment mass spectrometry, due to persistent iron interference. Despite >97% iron removal during ether extraction, the columns were unable to produce samples clean enough for precise mass spectrometry measurement, probably because iron was still plentiful enough to saturate the resin. The problem was corrected by using two clean, successive columns rather than one. Application of Microwave Digestion to Zinc Stable Isotope Analysis by Fast Atom Bombardment Mass Spectrometry. Data generated from tracer sampling can be used to quantitate and model zinc exchange between the plasma, red blood cells, and other compartmental pools. Although stable isotopes allow researchers to perform safe tracer experiments with special populations for whom radioisotopes are inappropriate, such as young children and pregnant women, the ability to monitor the tracer in the living body is more limited than that with radioisotopes. Given this limitation, red blood cells offer a convenient, readily accessible, and relatively inexpensive tissue for sampling. Two and one-half grams of red blood cells is sufficient sample size for determining zinc isotopic enrichment after intravenous administration of
