BIOCHEMICAL
MEDICINE
14, !8’&1%
(1975)
Copper in Erythrocytes by Flameless Atomic Absorption Spectroscopy WAYNE
B.
ROBBINS, AND
Depurrmenr
qf Chemistry.
BENJAMIN
JOSEPH UtliL,ersify
Received
A.
M.
of C‘inc~innuti. August
DEKOVEN.
CARUSO Cirfc~inrlrrti,
Ohio
IS?.?1
18, 197.5
INTRODUCTION
The advantages of flameless atomization in comparison with flame atomization in atomic absorption spectroscopy have been demonstrated repeatedly for a number of metals which have direct, or indirect physiological interest (l-3). However, the complex matrices normally encountered in physiological samples have presented a formidable problem to many workers, whether working with flame or flameless methods. This is evidenced by the many different sample preparation techniques found in the literature (3-6). Many of the procedures are of such length or detail that “same day” analysis of a sample with a complex matrix is difficult. This frequently means that the value of the final result in terms of clinical information may be diminished. This study describes a rapid method of sample preparation and determination of copper in red blood cells-a sample involving a complex matrix. With this technique an accurate micro-determination of trace metals such as copper can be made within an 8-hr shift with less than 4-hr total working time. EXPERIMENTAL
Apparatus The analyses were performed on a Jarrell-Ash 82-500 MVAA atomic absorption spectrometer (Waltham, Massachusetts). The instrument was operated in the absorption mode, and was peaked daily at the absorbing wavelength of 324.7 nm. A minimum damping setting of 1 was used with the coarse gain set as low as possible and no scale expansion. The constant radiation source was provided by a Tekmar (Cincinnati, Ohio) hollow cathode copper lamp operated at 7 mA. The standard burner head provided with the spectrometer was replaced with a Varian Techtron Model 63 Carbon Rod Atomizer (CRA, Melbourne, Australia). The support rods and graphite sampling chamber were situated in a stream of argon to minimize oxidation during the high temperature operation. The argon flow was
COPPER
IN
ERYTHROCYTES
185
regulated at 4.4 l/min by means of the gas control unit provided with the Carbon Rod Atomizer. The sampling chamber used with the atomizer was a hollow carbon tube (manufactured by Ultra Carbon Corporation, Bay City, Michigan). The tube was modified in our laboratories by drilling a hole in the inner surface 0.7 mm in depth and 1.4 mm in diameter. The tube was then repyrolyzed by passing a 1:3 mixture of methane/argon through the atomizer and carbon tube, while simultaneously firing the tube at a temperature of approx. 2000°C. This pyrolytic coating lasted a minimum of 75 to 80 firings before it was necessary to repyrolyze. A carbon tube may be repyrolyzed four or five times before a new tube is desirable. The atomizer was operated in the step atomization mode with the following voltage/time settings for copper: Dry-O.50 V/60 set; Ash-l .OOV/60 set; Atomize-6.70 VI2 sec. The output signals were monitored by measuring maximum peak deflections on a Hewlett Packard 7101B Chart Recorder (Palo Alto, California 94306) with a full scale deflection of less than 0.5 set (normal chart speed 0.5 in./min). The criteria for selection of the atomizer control and time settings follow: Dry Stage Voltage. A maximum voltage was selected so that the sample would dry in a minimum time without boiling and subsequent spattering. Ash Stage Voltage. The minimum voltage was used that could accomplish the following: Upon atomization with the recorder running at 2 in./min only one peak was observed which could not be further resolved; there was no decrease in peak height with further voltage increases without atomization of copper as compared to aqueous copper standards; and there was no observable background absorption at the nonabsorbing wavelength 3 19.4 nm. Time. During the ash cycle if the recorder indicated nonatomic absorption, the sample was ashed for an additional 30 set after the pen returned to baseline. Atomization Voltage. A minimum voltage was used so that additional samples of equal concentration when atomized yielded peak heights comparable with those obtained at higher voltages. Time. The minimum length of time that could meet the above criteria was selected. Samples, 5 ~1 in volume, were injected into the tube with a Pipetman (Rains Instrument Co., Boston, Massachusetts) &20~ 1 automatic pipette
186
ROBBINS,
DEKOVEN
AND
CARGSO
equipped with MLA (Medical Laboratory Automation. Inc.. Mt. Vernon. New York) disposable pipette tips. Decomposition bombs for destroying the organic matter have been previously described (7). All glassware was soaked a minimum of 16 hr in 33% HNQ followed by thoroughly rinsing first with distilled water and then with distilled water passed through a mixed bed ion-exchange resin (Crystalab Deeminizer, Cole Palmer, Chicago, Illinois). Materials Standard spike solutions were prepared daily from Fisher Brand Copper Atomic Absorption Standard (Fisher Scientific Co.. Pittsburgh. Pennsylvania). Methods Specimens were obtained from healthy male volunteer donors. They were drawn with Stylex disposable syringes (Pharmaseal Laboratories, Glendale, California) equipped with Monoject (Sherwood Medical Industries. Inc.. Delend, Florida) 22 gauge needles. Initial screening of needles and syringes indicated that there were no detectable copper contaminants. After acquisition, the specimens were immediately transferred into acid washed test tubes containing sodium citrate (prescreened for copper) at a concentration of 5 mg/ml of whole blood. The blood was centrifuged and the plasma was removed. The cells were washed once with prescreened 0.9% NaCl and then 0.5 to 0.6 ml of the cells were packed in graduated pipettes by centrifugation at 2500 rpm for 30 min. The red blood cells (RBCs) were then quantitatively transferred into a Teflon bomb insert and a 3 ml aliquot of redistilled HNQ, (G. Frederick Smith Chemical Co., Columbus, Ohio) was added. The bomb was sealed and incubated in an oven at 140°C for a minimum of 4 hr. After incubation, the bomb contents were diluted 50-fold. A total of six individuals were surveyed with multiple samples taken from each donor. Each sample was digested separately and the copper concentration determined by the method of standard addition described by Christian (8). RESULTS AND DISCUSSION Peak heights obtained were evaluated by a linear least squares FORTRAN computer program. The final data obtained from a minimum of two additions plus the original sample and a minimum of four injections for each addition are presented in Table 1. A typical recorder tracing and standard additions plot are depicted in Figs. I and 2. respectively. The peak in Fig. 1 labeled “Ash” is of particular interest. This peak results from destruction of residual organic residue during the Ash Cycle. Complete atomization of organic residue during this cycle is indicated by the
187
COPPER IN ERYTHROCYTES TABLE 1 ANALYTICAL DATA FOR COPPER IN ERYTHROCYTES
Specimen A
Sample 1 2 3 4
Picograms copper (5 fil injection)
Copper concentration” @&IO0 ml RBCs)
60.9 (3.0)” 65.8 (3.4) 55.2 (1.1) 68.2 (4.4)
122
Average 1 2 3 4
52.9 (2.2) 67.5 (1.4) 51.7 (2.1) 65.8 (5. I)
Average 1 2 3 4
126 117 t 8” 98
121 109 118
112 2 6” 63.9 72.8 64.0 58.4
(4.9) (24) (8.0) (0.4)
Average
107
128 116 108
115 + P
1
61.8 (8.2)
107
2 3
66.2 (8.7) 68.0 (5.3)
102
1 2 3
69.3 (4.4) 60.4 (22) 64.3 (8.9)
122 104 113 113 f 6”
53.0 (4.7) 63.0 (I .5)
91 107 99 + 8”
Average 1 2
105
105 -c 2”
Average
F
118 100
Average a Average for each sample is presented with mean deviation. h Extrapolated intercept on abscissa with computed standard deviation.
absence of any subsequent peak deflection above baseline noise at the nonabsorbing wavelength 319.4. This absence of “background” was determined for each sample. No corrections were necessary. The peak labeled “atomize” in Fig. 1 was reproducible from injection to injection within 1% transmittance. The results reported in Table 1 appear slightly higher than the range of 90-98 p.g/lOO ml that has been given by other workers (9, 10, ll), although statistically there is little meaningful difference. Considering the absolute picogram range in which this study was conducted (50-160 pg),
188
ROBBINS,
DEKOVEN
AND
CARUSO
AT0 MIZE
FIG.
I. A typical
recorder
tracing
representing
percentage
of absorption
the precision of the results is quite acceptable. Although this range is quite short, it is the most sensitive linear range for copper. Other workers described significant changes in sensitivity as a function of pH (3). It has been determined in this laboratory that as the pH of a sample was varied, the spreading characteristics of the sample on the tube changed significantly. It was observed that as the concentration of acid was increased, not only did the sample spread differently across the inner surface of the tube, but also the spreading patterns of same sample injections became erratic resulting in irreproducible atomization peak heights. This irreproducible spreading probably leads to variable concentrations of atomic vapor in the tube. As the sample spreads closer to either end of the tube, the concentration of atomic vapor appears to decrease as do the peak heights (12). The small indentation in the inner surface of the
13
FIG
2. A typical
standard
addition
plot.
COPPER
IN
ERYTHROCYTES
189
carbon tube, as described above, contains a sufficient amount of the 5 ~1 injection to insure more reproducible spreading and subsequent reproducible peak heights. The positioning of the depression directly under the sample injection port is the most likely position to insure reproducibility of injection. A primary consideration during the study was the small amount of sample necessary for analysis. Typically, 10 ml of blood were drawn from which 4 ml of packed cells were obtained. The method could easily be scaled down to use less than 0.5 ml of blood. This is an important factor when either this or any other trace metal analysis procedure is considered. Work completed in these laboratories indicate that the sample preparation method also is acceptable for blood serum, bovine liver, and freeze dried lung tissue (13). Combined with the sensitivities that flameless AA offers, the bomb digestion technique clearly increases the number of different samples that can be considered for analysis on a routine basis. SUMMARY
This study describes a method of analysis for copper in red blood cells. The sample is digested with nitric acid in a Teflon-lined steel bomb. It then is diluted and run without any further treatment. A small hole is drilled into the inner surface of the carbon tube to eliminate irreproducible sample spreading and subsequent irreproducible signals. An average value for the six persons surveyed is 110 pg/lOO ml. This method of sample preparation and analysis easily can be used to deal with other biological samples of complex matrix. ACKNOWLEDGMENTS The authors are grateful to the National Science Foundation and the National Institute for Occupational Safety and Health for support of the work by research grants GP 36478 and OH 0041.5, respectively. We also would like to express our appreciation to Providence Hospital (Cincinnati, Ohio) for the use of their facilities to obtain blood specimens.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 I.
Huffman, H. L., and Caruso, J. A., J. Agr. Food Chem. 22, 824 (1974). Evenson, M. A., and Warren, B. L., Clin. Chem. 21, 619 (1975). Evenson, M. A., and Anderson, Jr., C. T.. C/in. Chem. 21, 537 (1975). Himmelhoch. S. R., Sober, H. A., Vallee. B. L.. Peterson. E. A.. and Fuwa. K.. Biochemistry 5, 2523 (1966). Sunderman, F. W., and Roszel, N. 0.. Amer. J. C/in. Pnthol. 48, 286 (1967). Zachariasen. H.. Andersen, I.. Kostol, C., and Barton, R., C/in. Chem. 21, 562 (1975). Fricke, F. L., Rose, Jr., O., and Caruso, J. A., Talanra (in press). Christian, G. D., Anal. Chem., 41, 24A (1969). Blomfield. J., and MacMahon. R. A., J. C/in. Pdwl. 22, 136 (1969). Cartwright, G. E.. and Wintrobe. M. M.. Amer. J. C/in. Nurr. 14, 224 (1964). Burch, R. E.. Hahn, H. K. J.. and Sullivan, J. F., C/in. Chem. 21, 501 (1975).
190 I?. I?.
ROBBINS.
Sturgeon.
R. E.. Chakrabarti,
1240
(1975).
Robbins.
W.
B., Payne.
DEKOVEN
C. L., J. A..
DeKoven.
Maines.
AND
CARUSO
1. S.. and B. M..
and
Bertelb.
Caruw.
P. C.. Ad. I
4.. unpublished
(‘hc,n~. data.
47
MEDICINE
14, !8’&1%
(1975)
Copper in Erythrocytes by Flameless Atomic Absorption Spectroscopy WAYNE
B.
ROBBINS, AND
Depurrmenr
qf Chemistry.
BENJAMIN
JOSEPH UtliL,ersify
Received
A.
M.
of C‘inc~innuti. August
DEKOVEN.
CARUSO Cirfc~inrlrrti,
Ohio
IS?.?1
18, 197.5
INTRODUCTION
The advantages of flameless atomization in comparison with flame atomization in atomic absorption spectroscopy have been demonstrated repeatedly for a number of metals which have direct, or indirect physiological interest (l-3). However, the complex matrices normally encountered in physiological samples have presented a formidable problem to many workers, whether working with flame or flameless methods. This is evidenced by the many different sample preparation techniques found in the literature (3-6). Many of the procedures are of such length or detail that “same day” analysis of a sample with a complex matrix is difficult. This frequently means that the value of the final result in terms of clinical information may be diminished. This study describes a rapid method of sample preparation and determination of copper in red blood cells-a sample involving a complex matrix. With this technique an accurate micro-determination of trace metals such as copper can be made within an 8-hr shift with less than 4-hr total working time. EXPERIMENTAL
Apparatus The analyses were performed on a Jarrell-Ash 82-500 MVAA atomic absorption spectrometer (Waltham, Massachusetts). The instrument was operated in the absorption mode, and was peaked daily at the absorbing wavelength of 324.7 nm. A minimum damping setting of 1 was used with the coarse gain set as low as possible and no scale expansion. The constant radiation source was provided by a Tekmar (Cincinnati, Ohio) hollow cathode copper lamp operated at 7 mA. The standard burner head provided with the spectrometer was replaced with a Varian Techtron Model 63 Carbon Rod Atomizer (CRA, Melbourne, Australia). The support rods and graphite sampling chamber were situated in a stream of argon to minimize oxidation during the high temperature operation. The argon flow was
COPPER
IN
ERYTHROCYTES
185
regulated at 4.4 l/min by means of the gas control unit provided with the Carbon Rod Atomizer. The sampling chamber used with the atomizer was a hollow carbon tube (manufactured by Ultra Carbon Corporation, Bay City, Michigan). The tube was modified in our laboratories by drilling a hole in the inner surface 0.7 mm in depth and 1.4 mm in diameter. The tube was then repyrolyzed by passing a 1:3 mixture of methane/argon through the atomizer and carbon tube, while simultaneously firing the tube at a temperature of approx. 2000°C. This pyrolytic coating lasted a minimum of 75 to 80 firings before it was necessary to repyrolyze. A carbon tube may be repyrolyzed four or five times before a new tube is desirable. The atomizer was operated in the step atomization mode with the following voltage/time settings for copper: Dry-O.50 V/60 set; Ash-l .OOV/60 set; Atomize-6.70 VI2 sec. The output signals were monitored by measuring maximum peak deflections on a Hewlett Packard 7101B Chart Recorder (Palo Alto, California 94306) with a full scale deflection of less than 0.5 set (normal chart speed 0.5 in./min). The criteria for selection of the atomizer control and time settings follow: Dry Stage Voltage. A maximum voltage was selected so that the sample would dry in a minimum time without boiling and subsequent spattering. Ash Stage Voltage. The minimum voltage was used that could accomplish the following: Upon atomization with the recorder running at 2 in./min only one peak was observed which could not be further resolved; there was no decrease in peak height with further voltage increases without atomization of copper as compared to aqueous copper standards; and there was no observable background absorption at the nonabsorbing wavelength 3 19.4 nm. Time. During the ash cycle if the recorder indicated nonatomic absorption, the sample was ashed for an additional 30 set after the pen returned to baseline. Atomization Voltage. A minimum voltage was used so that additional samples of equal concentration when atomized yielded peak heights comparable with those obtained at higher voltages. Time. The minimum length of time that could meet the above criteria was selected. Samples, 5 ~1 in volume, were injected into the tube with a Pipetman (Rains Instrument Co., Boston, Massachusetts) &20~ 1 automatic pipette
186
ROBBINS,
DEKOVEN
AND
CARGSO
equipped with MLA (Medical Laboratory Automation. Inc.. Mt. Vernon. New York) disposable pipette tips. Decomposition bombs for destroying the organic matter have been previously described (7). All glassware was soaked a minimum of 16 hr in 33% HNQ followed by thoroughly rinsing first with distilled water and then with distilled water passed through a mixed bed ion-exchange resin (Crystalab Deeminizer, Cole Palmer, Chicago, Illinois). Materials Standard spike solutions were prepared daily from Fisher Brand Copper Atomic Absorption Standard (Fisher Scientific Co.. Pittsburgh. Pennsylvania). Methods Specimens were obtained from healthy male volunteer donors. They were drawn with Stylex disposable syringes (Pharmaseal Laboratories, Glendale, California) equipped with Monoject (Sherwood Medical Industries. Inc.. Delend, Florida) 22 gauge needles. Initial screening of needles and syringes indicated that there were no detectable copper contaminants. After acquisition, the specimens were immediately transferred into acid washed test tubes containing sodium citrate (prescreened for copper) at a concentration of 5 mg/ml of whole blood. The blood was centrifuged and the plasma was removed. The cells were washed once with prescreened 0.9% NaCl and then 0.5 to 0.6 ml of the cells were packed in graduated pipettes by centrifugation at 2500 rpm for 30 min. The red blood cells (RBCs) were then quantitatively transferred into a Teflon bomb insert and a 3 ml aliquot of redistilled HNQ, (G. Frederick Smith Chemical Co., Columbus, Ohio) was added. The bomb was sealed and incubated in an oven at 140°C for a minimum of 4 hr. After incubation, the bomb contents were diluted 50-fold. A total of six individuals were surveyed with multiple samples taken from each donor. Each sample was digested separately and the copper concentration determined by the method of standard addition described by Christian (8). RESULTS AND DISCUSSION Peak heights obtained were evaluated by a linear least squares FORTRAN computer program. The final data obtained from a minimum of two additions plus the original sample and a minimum of four injections for each addition are presented in Table 1. A typical recorder tracing and standard additions plot are depicted in Figs. I and 2. respectively. The peak in Fig. 1 labeled “Ash” is of particular interest. This peak results from destruction of residual organic residue during the Ash Cycle. Complete atomization of organic residue during this cycle is indicated by the
187
COPPER IN ERYTHROCYTES TABLE 1 ANALYTICAL DATA FOR COPPER IN ERYTHROCYTES
Specimen A
Sample 1 2 3 4
Picograms copper (5 fil injection)
Copper concentration” @&IO0 ml RBCs)
60.9 (3.0)” 65.8 (3.4) 55.2 (1.1) 68.2 (4.4)
122
Average 1 2 3 4
52.9 (2.2) 67.5 (1.4) 51.7 (2.1) 65.8 (5. I)
Average 1 2 3 4
126 117 t 8” 98
121 109 118
112 2 6” 63.9 72.8 64.0 58.4
(4.9) (24) (8.0) (0.4)
Average
107
128 116 108
115 + P
1
61.8 (8.2)
107
2 3
66.2 (8.7) 68.0 (5.3)
102
1 2 3
69.3 (4.4) 60.4 (22) 64.3 (8.9)
122 104 113 113 f 6”
53.0 (4.7) 63.0 (I .5)
91 107 99 + 8”
Average 1 2
105
105 -c 2”
Average
F
118 100
Average a Average for each sample is presented with mean deviation. h Extrapolated intercept on abscissa with computed standard deviation.
absence of any subsequent peak deflection above baseline noise at the nonabsorbing wavelength 319.4. This absence of “background” was determined for each sample. No corrections were necessary. The peak labeled “atomize” in Fig. 1 was reproducible from injection to injection within 1% transmittance. The results reported in Table 1 appear slightly higher than the range of 90-98 p.g/lOO ml that has been given by other workers (9, 10, ll), although statistically there is little meaningful difference. Considering the absolute picogram range in which this study was conducted (50-160 pg),
188
ROBBINS,
DEKOVEN
AND
CARUSO
AT0 MIZE
FIG.
I. A typical
recorder
tracing
representing
percentage
of absorption
the precision of the results is quite acceptable. Although this range is quite short, it is the most sensitive linear range for copper. Other workers described significant changes in sensitivity as a function of pH (3). It has been determined in this laboratory that as the pH of a sample was varied, the spreading characteristics of the sample on the tube changed significantly. It was observed that as the concentration of acid was increased, not only did the sample spread differently across the inner surface of the tube, but also the spreading patterns of same sample injections became erratic resulting in irreproducible atomization peak heights. This irreproducible spreading probably leads to variable concentrations of atomic vapor in the tube. As the sample spreads closer to either end of the tube, the concentration of atomic vapor appears to decrease as do the peak heights (12). The small indentation in the inner surface of the
13
FIG
2. A typical
standard
addition
plot.
COPPER
IN
ERYTHROCYTES
189
carbon tube, as described above, contains a sufficient amount of the 5 ~1 injection to insure more reproducible spreading and subsequent reproducible peak heights. The positioning of the depression directly under the sample injection port is the most likely position to insure reproducibility of injection. A primary consideration during the study was the small amount of sample necessary for analysis. Typically, 10 ml of blood were drawn from which 4 ml of packed cells were obtained. The method could easily be scaled down to use less than 0.5 ml of blood. This is an important factor when either this or any other trace metal analysis procedure is considered. Work completed in these laboratories indicate that the sample preparation method also is acceptable for blood serum, bovine liver, and freeze dried lung tissue (13). Combined with the sensitivities that flameless AA offers, the bomb digestion technique clearly increases the number of different samples that can be considered for analysis on a routine basis. SUMMARY
This study describes a method of analysis for copper in red blood cells. The sample is digested with nitric acid in a Teflon-lined steel bomb. It then is diluted and run without any further treatment. A small hole is drilled into the inner surface of the carbon tube to eliminate irreproducible sample spreading and subsequent irreproducible signals. An average value for the six persons surveyed is 110 pg/lOO ml. This method of sample preparation and analysis easily can be used to deal with other biological samples of complex matrix. ACKNOWLEDGMENTS The authors are grateful to the National Science Foundation and the National Institute for Occupational Safety and Health for support of the work by research grants GP 36478 and OH 0041.5, respectively. We also would like to express our appreciation to Providence Hospital (Cincinnati, Ohio) for the use of their facilities to obtain blood specimens.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 I.
Huffman, H. L., and Caruso, J. A., J. Agr. Food Chem. 22, 824 (1974). Evenson, M. A., and Warren, B. L., Clin. Chem. 21, 619 (1975). Evenson, M. A., and Anderson, Jr., C. T.. C/in. Chem. 21, 537 (1975). Himmelhoch. S. R., Sober, H. A., Vallee. B. L.. Peterson. E. A.. and Fuwa. K.. Biochemistry 5, 2523 (1966). Sunderman, F. W., and Roszel, N. 0.. Amer. J. C/in. Pnthol. 48, 286 (1967). Zachariasen. H.. Andersen, I.. Kostol, C., and Barton, R., C/in. Chem. 21, 562 (1975). Fricke, F. L., Rose, Jr., O., and Caruso, J. A., Talanra (in press). Christian, G. D., Anal. Chem., 41, 24A (1969). Blomfield. J., and MacMahon. R. A., J. C/in. Pdwl. 22, 136 (1969). Cartwright, G. E.. and Wintrobe. M. M.. Amer. J. C/in. Nurr. 14, 224 (1964). Burch, R. E.. Hahn, H. K. J.. and Sullivan, J. F., C/in. Chem. 21, 501 (1975).
190 I?. I?.
ROBBINS.
Sturgeon.
R. E.. Chakrabarti,
1240
(1975).
Robbins.
W.
B., Payne.
DEKOVEN
C. L., J. A..
DeKoven.
Maines.
AND
CARUSO
1. S.. and B. M..
and
Bertelb.
Caruw.
P. C.. Ad. I
4.. unpublished
(‘hc,n~. data.
47
