Simultaneous Determination of Calcium, Copper, Manganese, and Magnesium in Serum by Neutron Activation Analysis SIR: A n understanding of the role of metalloenzymes in the normal and pathological metabolic pattern requires an accurate analysis of trace elements in serum. Neutron activation analysis combines the inherent sensitivity required for such a program with easy adaptability to multiple element analysis. Although most of the previous radiochemical determinations in biological samples have involved single element isolation, simultaneous quantitative analysis should be readily achievable provided interfering sodium and chlorine activities are removed. A solvent extraction procedure, in which the elements to be determined are first removed from the bulk of the contaminants, seemed a promising approach. Thenoyltrifluoroacetone (TTA) was chosen as the chelating agent since it is known to extract copper and calcium (S), to form a magnesium chelate insoluble in benzene ( 4 ) , and a t least partially to extract manganese ( 2 ) . 4 t a constant pH of 9.0, a mixed solvent system of TTA in benzene-tetrahydrofuran was used to separate the desired elements. n’eutron activation, isolation of the elements, and gamma ray spectrometry followed. The accuracy of the method was checked by extracting known chemical solutions. Applicability to biological samples was tested with pooled serum samples. EXPERIMENTAL
Apparatus. Irradiations were performed in a Triga Mark I reactor a t a flux of 1.1 X 10” n/cm.2/sec. The gamma activities were measured with a 400-channel pulse height analyzer equipped with a 2 X 2 inch NaI (Tl) crystal. Reagents. Standard solutions of the elements studied were prepared from spectrographically pure compounds (Johnson Matthey Co., Ltd., London, England) in dilute HNOa. A TTA solution (1%)was prepared in a 60/40 (v./v.) mixture of benzene (99 mole yo, Fisher Scientific Co.)/tetrahydrofuran (THF). The buffer solution of pH 9.0 was made from reagent grade NH40H and glacial acetic acid. A11 other reagents were prepared from reagent grade chemicals in demineralized water. Procedure. BLOODSERUM. The large pooled sample was obtained from the Nebraska State Health Laboratory, and the smaller pooled samples were received from the Clinical Laboratory a t the Hospital. No precautions were taken t o avoid elemental contamination by the collectors for the State Health Laboratory. Acid
washed glassware and disposable needles with plastic hubs were used by the hospital laboratory. PROTEINPRECIPITATION. To 5-ml. aliquots of the pooled serum samples in 40-ml. centrifuge tubes, 5 ml. of hot 4y0 picric acid were added and the mixture was shaken well. Centrifugation a t 2000 r.p.m. for 20 minutes was followed by shaking the samples and centrifuging another 20 minutes. A 5-ml. aliquot of the supernatant, corresponding to 2.5 ml. of serum, was removed for the chemical separation. PRE-IRRADIATION SEPARATION. The solution to be separated was pipetted into a 60-ml. separatory funnel. One drop of 0.1% brilliant yellow and 5 ml. of 1% TTA in 60/40 benaene/THF were added. The pH was adjusted to the color change with 0.5N NHaOH, and 3 mi. of pH 9.0 buffer added. The mixture was shaken for 3 minutes, the layers were allowed to separate, the aqueous layer was discarded, and the organic solution was washed with 3 ml. of pH 9.0 buffer. The buffer was withdrawn, 4 ml. of 1 3 HKO:, were added to revert the metals to the aqueous phase and the mixture was shaken for 3 minutes. With separation of the phases, the aqueous solution of metal ions was drained into a vycor vial which was sealed in a polyethylene bag and placed in a polyethylene rabbit for irradiation. IRR-4DIATION PROCEDURE. Rotation of the sample a t 1 r.p.m. during the 1-hour irradiation ensured a uniform flux throughout positions in the “Lazy Susan.” The flux was determined by a gold wire activation. POST-IRRADISTION TREATMENT. After irradiation, the metal ions were re-extracted into the organic phase using the same quantities of reagents as in the preirradiation separation. A 3-ml. aliquot, 75% of the irradiated sample, was pipetted into a separatory funnel which contained brilliant yellow and TTA in benzene/THF. After adjustment of the pH and addition of buffer, the mixture was shaken for 3 minutes. With separation of the layers, a 4-ml. aliquot of the organic phase, 60% of the original ir-
radiated sample, was transferred to a cold polyethylene vial for counting. COUNTING PROCEDURE. Each sample was counted twice for 10 minutes live time, a t 10 minutes and 100 minutes after removal from the reactor. SEPARATION OF STANDARDS.A library of standards was obtained by irradiating the stock solutions of each element, diluting the irradiated concentrated standard and transferring 4 ml. to a cold vial for counting. The irradiation and counting procedures were the same as those employed in the samples. REAGENT BLANKS.The average spectrum of six reagent blanks was subtracted from the individual sample spectra. CALCULATIONS. The concentrations of the elements were determined by 2 sets of 2 simultaneous linear equations using the IBM 1620 ( I ) . The first two equations in two unknowns solved the 100 minute spectrum for Cu and Mn content. At 100 minutes after removal from the reactor the Illgn and Ca49,which had decayed through more than 10 half-lives, contributed no significant data and a spectrum essentially due to Cue4 and M n s remained. The summation of counts in the 0.51-hl.e.v. area, or channels 45-60, was used for the Cu determination and channels 80-96, the integration of the 0.845 M.e.v. peak, were used in analyzing >In. To calculate Mn and Cu contributions a t 10 minutes after activation, the following simple equation was used : Mass of X in sample (10-min. X std.) = Mass of X in std. 10-min. X contribution where X
=
element
Subtraction of the derived 10-minute Cu and Mn contributions from the actual 10-minute count left a remaining spectrum due to MgZ7and Ca49. Using two equations in two unknowns, the h9g (channels 80-96) and Ca (channels 240-279) contents were determined.
Table 1.
Analysis of Solution of Known Chemical Composition Mn, pg./ml. Cu, pg./ml. Ca, mg./ml. Mg, mg./ml. Solution No. 1, Ar = 17 Mean 0.033 3.32 0.0930 0.0214 Std. dev. 0.001 0.11 0.0038 0.0011 Weighed amt. 0.033 3.33 0.1070 0.0214 Found 0,033 3.32 0.0930 0.0214 Yield, yo 100 100 87 100 Solution No. 2, N = 11 Mean 0.030 2.93 0.0927 0.0327 Std. dev. 0.003 0.07 0.0023 0.0029 Weighed amt. 0.030 3.13 0.1025 0.0310 Found 0.030 2.93 0.0927 0.0327 Yield, yo 100 94 91 105
VOL. 38, NO. 1, JANUARY 1966
141
RESULTS
Before actual serum samples were analyzed, two solutions containing known amounts of Ca, Cu, Mn, and Mg in the presence of interfering ions in their amroximate serum concentrations (K+, Na+, C1-, Br-, I-, Hg+2,As+3, Fe+2, Fef3, and Zn+2) were quantitatively determined. Results are presented in Table I. From the analysis of the solutions, it appeared that only approximately 90% of the Ca present in the sample was extracted; this is confirmed in both the results of known chemical solutions and the recovery tests performed by adding known amounts of Ca to the supernatants. If aqueous standards are used as described previously, the Ca value obtained through the analysis should be corrected for only 90% extraction. The accuracy of the procedure, after adjustment of the Ca values, was 96 to 100%; the precision as determined by one standard deviation was about *6%. The proteins of aliquots of a pooled serum sample from the State Health Laboratory were precipitated and the Ca, Mn, Mg, and Cu content of the supernatant was determined. The results of these 21 analyses are shown in Table 11. The standard deviation of the method on various aliquots of serum was about 10% for Ca, Cu, and hfg, with Mn being about 16%. Table I1 also gives the results of replicate analyses on two pooled serum samples obtained from the Hospital Clinical Laboratory.
1.00
PkV
Figure 1.
Resolution of spectrum
-- Actual serum spectrum after separation -Mathematical composite of individual quantities Results of recovery tests are in Table 111. Known quantities of each of the four elements were added to the supernatant of the serum samples. The Ca recovery tests are corrected for 90% extraction. DISCUSSION
Standard spectra for each of the elements were obtained prior to activa -
Table II. Analysis of Pooled Serum
Mn, gg./ml. State Health Laboratory N Mean Std. dev. Hospital Laboratory No. 1, N = 4 Mean Std. dev. No. 2, N = 3 Mean Std. dev.
Mn, Pg." cu, rg. Ca, mg. Mg, mg.
Mg, mg./ml.
0,019 0.003
1.43 0.12
0.1199 0.0092
0.037 0,004
0.010 0.001
1.34 0.03
0.089 0.006
0.020 0.000
0.012 0.001
1.27 0.02
0.116 0.004
0.029 0.000
Recovery of Mn, Cu, Ca, and Mg
Added
Theo.
0.027 0.050 3.35 3.20 0.2265 0.2995 0,051 0.093
0.060 0.060 6.26 6.26 0.2050 0.2050 0.062 0.062
0.087 0.110 9.61 9.46 0.4315 0.5045 0.113 0.155
All values in totals. Triplicate analysis. c Duplicate analysis.
0
Ca, mg./ml.
Normal
0
142
Cu, pg./ml.
= 23
Table 111.
ANALYTICAL CHEMISTRY
2.80
Found 0.078 0.099 9.47 8.38 0.4154 0.4935 0.106 0.166
Yield, % 89b 9oc 99b 89 99b 9gC 94b 107"
tion of the samples. Routine analysis could thus be performed using a comparative method and computing a daily flux. If volumes of solutions, irradiation time, decay time, and counting time were kept constant, all samples could be analyzed using the initial standards. Aqueous standards, standards not extracted through the chemical separation, were used for two reasons. First, by using these standards as a reference, the chemical yields could be determined. Second, contamination of the standards from reagents was eliminated. Although other metal ions were extracted a t pH 9.0 with TTX-e.g. Zn+2, Hg+2-after a 1-hour irradiation the concentrations, cross sections, and halflives of such elements caused their activity to be negligible in the presence of Ca, Cu, Mn, and Xg. That this is so is shown in Figure 1. The dotted line is the actual spectrum obtained in a separation of Ca, Cu, Mn, and Mg from serum a t 10 minutes after activation. The solid line is a mathematical reconstruction obtained by summing the contributions of each element to the entire spectrum; that is, upon solution of the simultaneous equations, the ratio of the mass of the element to the mass of the standard was multiplied by the respective standard spectrum. The summation of these individual spectra allowed a check on the mathematical analysis of the original sample. Since the two spectra are essentially identical except for standard deviations due to count rate, it can be stated that the four elements were present and in the quantities computed. The liquid-liquid extraction was per-
formed before irradiation. The preirradiation separation offered two advantages. The absence of a lengthy post-irradiation separation allowed the short half-lived magnesium-27 and calcium-49 to be determined and the radioactive samples could be handled with tracer techniques because of the low activity generated by irradiation of the metal ions. The reversion of the metal chelates to the acid solution before irradiation and the subsequent formation of the chelates after activation lowered the activity of the blank and decreased possibilities of contamination from K a and C1 in the irradiation container. By taking aliquots from the reversion and back extraction, the procedure was kept quantitative. The correction factor for the 60% of the original sample actually counted was also programmed into the computer. The values obtained from the pooled serum samples are not meant to be
normal values, but rather show the feasibility and reproducibility of the method in actual biological samples. The samples obtained from the Clinical Laboratory were consistently lower in Ca, Cu, Mn, and Mg concentration. There is a definite possibility that the first pooled serum in Table I1 was contaminated because the blood had been collected from all parts of the state and knowledge of techniques in collection was impossible to obtain. Despite this fact, the values of Cu and Mn did fall within the normal range in the literature, although the N g and Ca contents were slightly higher. The relative simplicity of a simultaneous determination as opposed to single element analyses renders this method practical, easy, and convenient by comparison. The accuracy of the method and the precision are equivalent to, and in most cases surpass, the best methods nom available for single element activation analysis of serum.
ACKNOWLEDGMENT
The authors thank R. E. Ogborn for his support of this work, A. J. Blotcky for the use of the reactor service, and B. T. Watson and F. J. Kerrigan for data processing. LITERATURE CITED
(1) Blotcky, A. J., Watson, B. T., Ogborn,
R. E., Proceedings, Applications of Computers to Nuclear and Radiochemistry, Gatlinburg, Tenn., 1962. (2) Khopkar, S. RI., De, A. J., Z. Analyt. Chem. 171. 241 (1959). (3) Moore, F. L., Am. fioc. Testing Mater., 238, 13 (1958). (4) Poskanzer, A. &I., Foreman, B. M., Jr., J . Znorg. Nucl. Chem. 16, 323 (1961).
RI. C. HAVEN G. T. HAVEN A. L. DUNN
Special Laboratory of Nuclear Medicine and Biology Veterans Administration Hospital Omaha, Neb.
Re-evaluation of the Potential of the Tris(4,7-Dimethyl-l,10-
Phe na nt hroline)iron (I I I)-Tris( 4,7-Di met hy 1-1/I 0-Phena nt hroline)iron(II) Couple in Water SIR: In two previous articles from this laboratory on the use of the halfwave potential for the oxidation of tris(4,7-dimethyl- 1,lo-phenanthro1ine)iron(I1) [4,7-DMPFe(II)] in various media for the evaluation of differences in liquid-junction potentials (S), we reported a value of +0.830 volt vs. S.C.E. for this couple in aqueous 0.10M lithium perchlorate solution. Because the potential of this redox system in water is of considerable importance in assessing solvent effects on redox systems in nonaqueous media, we should like to report the following information. Recently, it was brought to our attention that our previously reported value for the half-wave potential for the oxidation of 4,7-DMPFe(II) in water, obtained by extrapolation using mixed solvents of varying water content, could be in error ( 2 ) . [The extrapolation method was used for the evahation of the potential of the 4,7-DMPFe(III)4,7-DMPFe(II) redox pair in water because of the insolubility of 4,7DMPFe(I1) perchlorate in this solvent. The validity of this method has been checked with a number of systems in which the half-wave potentials of the couple were known.] We, therefore, re-evaluated the potential of this system in water by the same extrapolation method, The half-wave potentials obtained for the couple in acetonitrile-
water mixtures of 50 (1-0.800 volt), ricinium ion-ferrocene couples, 70 ($0.758 volt), and 80 (+0.734 respectively. The new value for the volt) volume yo water and acetone4,7 - DMPFe(II1)-4,7 - DMPFe(I1) water mixtures of 0 (+0.925 volt), couple is also in close agreement with the 50 (+0.808 volt), and 70 (+0.767 volt) value of +0.64 volt us. S.C.E. (+0.88 volume yo water were extrapolated to volt us. J reported by Brandt 100% water by the use of EIi2 vs. and Smith(1) for the couple in aqueous volume % water plots. Both extrap0.1M sulfuric acid solution. The olations gave $0.695 volt us. S.C.E. potential of the same couple in ethanol for the potential of this redox system in also was checked using acetonitrileaqueous 0.10JI lithium perchlorate. ethanol mixtures. The extrapolated The +0.830-volt value reported prepotential in this case is in reasonably viously, therefore, is in error. No good agreement with the previously explanation can be offered a t this time reported value. for the completely different potential ACKNOWLEDGMENT values which were obtained previously for the acetonitrile-water mixtures. We thank Professor Kolthoff for With this new potential for the 4,7sending us a copy of his paper in advance DMPFe(III)-4,7-DR;IPFe(II) couple in of publication. water, we calculate a value of +0.165 for the difference in liquid-junction LITERATURE CITED potential between 0.10X LiC104 in (1) Brandt, W. W., Smith, G. F., ANAL. acetonitrile vs. aqueous S.C.E. and CHEM.21, 1313 (1949). 0.10M LiC104 in water vs. aqueous (2) Kolthoff, I. M., Thomas, F. G., J . S.C.E. (Eliz acetonitrile = +0.860 volt Phys. Chem. 69, 3049 (1965). V S . S.C.E., Eli2 HzO = f0.695 volt. US. (3) Nelson, I. V., Iwamoto, R. T., ANAL. S.C.E.) This A E I , , , , ~ . ,value ~ ~ ~ ~is , ~ ~ CHEM.33, 1795 (1961); 35, 867 (1963). consistent with the values of -1-0.157 FLOYD FARHA, JR. volt and +0.220 volt obtained by REYNOLD T. IWAMOTO Kolthoff and Thomas (2) for the liquidDepartment of Chemistry junction potential between acetonitrile University of Kansas containing (C2Hj)4NC104 in 0.10Jf conLawrence, Kan. centration and aqueous S.C.E. using the WORE supported by the Directorate of tris ( 1 , l O - phenanthroline) iron(II1)-trisChemical Sciences, Air Force Office of (1,lO-phenanthroline) iron (11) and ferScientific Research, Grant No. 220-63. VOL. 38, NO. 1, JANUARY 1966
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