MICROCHEMICAL Sensitive JOURNAL 16, 304-310 (1971) Microreacting RUTH Ligand WATKINS for Serum Copper AND BENNIE ’ ZAK Department of Pathology, Wayne State University, School of Medicine and Detroit General Hospital, Detroit, Michigan 48207. Received January 25, 1971 Several compounds have proven useful as reagents for the determination of copper contained in biological sources. Commonly suggested ligands are neocuproine (10)) bathocuproine and its sulfonate (7, 11), diethyldithiocarbamate (8, 15)) dibenzyldithiocarbamate (3)) biscyclohexanoneoxalyldihydrazone (,6), oxalyldihydrazide plus acetaldehyde (I, 9, 12, 14), dithizone (2), and 1,5-diphenylcarbohydrazide (5). The molar absorptivities reported for their complexes of copper ranged from 7950 for neocuproine to 58,500 for 1,5-diphenylcarbohydrazide. Recently a reagent of excellent sensitivity was proposed for the determination of copper in tissues and body fluids (4). The use of the compound, N,N,N1,N1-tetraethylthiuram disulfide, also called disulfiram and Antabuse, was described for a procedure in which total destruction by wet ashing with perchloric acid (PCA) was employed. A PCA filtrate technique was also suggested for serum copper but procedural details were limited, making it difficult to follow the authors’ exact intent. In that study (4), it was pointed out that the use of trichloroacetic acid (TCA) and HCl to prepare a filtrate proved unsatisfactory and troublesome but just why this should be so was not considered or discussed. When an attempt was made to use TCA filtrates of serums and the suggested solution of the color reagent, turbidities developed which were indeed troublesome. A subsequent follow-up study of the turbidity showed it to be due to precipitation of the disulmam reagent. The addition of a solubilizing solvent such as ethanol cleared the solution but the dilution required created a new problem, i.e., loss of sensitivity. Finally, after testing several substitute solvents, glacial acetic acid was chosen as the reagent medium for this system. By this simple expedient of changing the solvent, characteristics of the color reagent turbid solutions were not encountered and the new reagent could be 1 Supported in part by grant-in-aid Corporation. from Detroit 304 General Hospital Research SERUM COPPER 305 used to obtain relatively high absorbance values with concentrated TCA filtrates prepared from 1: 1 ratios of serum to precipitating agent. The present report describes a simplified procedure for serum involving only two chemical steps, one of which is the conventional preparation of a TCA filtrate and includes a spectrophotometric study of the characteristics of the sensitive reaction. EXPERIMENTAL METHODS Reagents Stock trichloroacetic acid solution, 100 g/dl. Stock copper standard solution, 50 &ml. Working copper standard solutions. Pipet 0.0, 1.0, 2.0, and 3.0 ml of the stock copper standard into volumetric flasks. To each add 10 ml of the stock TCA solution, and dilute them to the mark of the flask with metal-free H,O and mix them well. N,N,N1,N1-Tetraethylthiuram d&u&de. Dissolve 40 mg of the compound to 1 dl with glacial acetic acid. Trichloroacetic acid (TCA)-precipitating agent. Dilute 20 ml of the stock TCA solution to 1 dl with metal-free distilled H?O. Procedure Pipet 2.0 ml of the TCA precipitating agent into 2 ml of sample, and mix thoroughly. Centrifuge out the precipitate and remove a 2-ml aliquot into a l-cm cuvette. Add 0.5 ml of color reagent, mix the solution well, and wait 5 minutes. Measure the absorbance against a reagent blank at 425 nm. Standardization Treat 2-ml aliquots of each working copper standard with 0.5 ml of color reagent, wait 5 minutes, and measure the absorbance of the standards against the blank at 425 nm. These 1: 1 filtratelike solutions of standards contain 0, 50, 100, and 150 pg/dl but correspond to serum values which would be prepared from an original 0, 100, 200, and 300 pg/dl. DISCUSSION The molar absorptivity for copper in the reaction with N,N,Nl,Nltetraethylthiuram disulfide (disulfiram) is stated to be 37,000 when the reaction medium was a strong perchloric acid solution in which the color reagent added was dissolved in acetone and water (4). When the reagent dissolved in glacial acetic acid was added to a trichloroacetic acid filtrate as described under Procedure, the molar absorptivity was 306 WATKINS AND ZAK 33,300, 11% less than reported for the stronger acid medium. However, the small loss in molar absorptivity still results in an extremely sensitive copper reaction. In addition, it enables one to use a buffered portion or portions of the same filtrate for the determination of iron or zinc if either or both were desired ( 13). If the stronger acid filtrate were used, buffering would involve greater dilution and lower absorbance values. There is a structural resemblance between N,N,NI,N’-tetraethylthiuram disulfide, disulfide I, and diethyldithiocarbamate II. The spectrum of the copper complex of II is shifted hypsochromically from 440 to 425 nm for II. W, \ N-C=S CA S-Na ‘N-6 C,H! 0) \\ S C,H/ 4 C,JL I 7 \N-C=S (II) In view of the structural similarities, one might expect some similarities in chemical reactivity. It has been proven that II is not interfered with by any of the ions normally present in serum even when their concentrations were far in excessof normal limits (4). But when diethyldithiocarbamate is used as a copper reagent it requires a masking reagent for iron or else the results for copper will be spurious. Therefore, the effect of iron on the suggested reaction was studied in the following manner. A copper solution was treated as described under Procedure, a spectrum was drawn between the wavelengths of 550 and 350 nm and it is shown as curve 1 in Fig. 1. When this same amount of copper was mixed with 1 mg of iron, the reaction carried out and the spectrum drawn as before, an apparently undistorted curve for copper was obtained (Curve 2, Fig. 1) but the peak intensity was increased by about 13%. A third solution containing only 1 mg of iron but no copper was scanned in order to establish or rule out copper contamination of the iron added and the results of this reaction are also graphed (Curve 4) in Fig. 1. On observing these spectra the inference can be drawn that the reaction sensitivity for copper appears to be enhanced by the presence of iron even though iron itself does not react with the ligand. Zinc is another trace metal of serum whose concentration is normally similar to those of copper and iron. Therefore, its effect on the copper reaction was tested as with iron and the graphic result is shown as Curve 3 in SERUM COPPER 307 FIG. 1. Shows the augmenting effect of iron and zinc on the reaction of copper with the color reagent: (1) the spectrum for copper alone; (2) the spectrum when the copper is contaminated with 1 mg of iron/dl; (3) the spectrum for iron alone; (4) the spectrum when the copper is contaminated with 1 mg/dl of zinc; and (5) the spectrum for zinc alone. All spectra shown were displaced 50 nm so that they can be seen more easily. Fig. 1. The augmenting effect of 1 mg of zinc on 300 pg of copper is much less, only about 3%. Zinc, like iron does not undergo reaction with the reagent at all as shown in Curve 5, and this seems to rule out copper contamination as the reason for color augmentation. Since the error for this large concentration of zinc is small, it would seem safe to ignore zinc as a contaminant of any importance in the reaction and it was studied no further. However, in serum, the iron concentrations encountered are usually lower than the copper concentrations. To study the reaction, a series of copper standards were reacted with the reagent in the absence and presence of several concentrations of iron and the spectra of the resulting colored solutions are shown in Fig. 2. At these ranges of concentrations for the two metals, there does not appear to be a significant error from the iron present, and for all practical purposes it seems feasible to ignore iron as a contaminant of any real importance. The 308 WATKINS AND ZAK FIG. 2. Shows the spectra of 100, 200, and 300 pg/dl of copper alone, and then when each is contaminated with 50, 100, 150, and 200 ,ug/dl of iron. Each spectral group was displaced 50 nm so that they can be seen more easily. study described in Fig. 2 can also be used to indicate the precision one could expect to encounter for the color reaction itself. The results indicated in addition to subsequent recoveries made at other times show a calculated standard deviation of AZ 4.56 throughout the range tested. Table 1 shows the means, ranges, and standard deviations obtained for three different concentrations of copper using the described procedure. The amount of ligand required in the reaction along with the time TABLE 1 RECOVERIES OF KNOWN CONCENTRATIONS OF COPPER WITH RANGES, MEANS AND STANDARD DEVIATIONS Samples Present a 12 10 12 100 200 300 a Copper cone (Mg/dl). Mean zt-1 SD 95-108 101 194-208 293-309 203 299 3.7 4.4 4.8 Range found SERUM COPPER 309 FIG. 3. Shows time and reagent concentration graphs when solutions containing 4, 8, 12, 20, and 40 mg of disulfiram/dl of glacial acetic acid are reacted with a fixed quantity of copper and the developing absorbances followed with time. The base lines were shifted slightly in absorbance so that plateaus can be seen more easily and the times were shifted by 5 minutes for the same reason. required to achieve full color formation for that amount is graphically described in Fig. 3. Different amounts of N,N,W,W-tetraethylthiuram disulfide, 4, 8, 12, 20, and 40 mg/dl, were added to a 300 pg/dl solution of copper and the absorbances continually graphed for 30-minute periods at 425 nm. The curves of Fig. 3 show that peak absorbance is reached quicker as the concentration of ligand is increased with the plateau for the time factor ranging between 20 and 40 mg/dl. Obviously, the amount described for use under Reagents is optimal for the reaction. As shown, the color formed is quite stable for the 30-minute period in which the continuous drawings were made and in fact is stable for several times longer than that time period. SUMMARY A rapid and simple filtrate procedure for the determination of serum copper is described. It involves only 2 chemical steps and is quite sensitive with a molar absorptivity of 33,300. The color formed is stable for more than 30 minutes and the results indicate that the reaction is quite applicable to the determination of copper found in serum. REFERENCES I. Beale, R. N., and Croft, D., The microdetermination of biological with oxalyldihydrazide. J. Clin. Puthol. 17, 260-263 (1964). copper 310 WATKINS AND ZAK 2. Butler, E. J., and Newman, G. E., An absorptiometric method for the determination of traces of copper in biological materials with dithizone. Clin. Chim. Acra 11, 452-460 (1965). 3. Georgio, A. J., Cartwright, G. E., and Wintrobe, M. M., Determination of urinary copper by means of direct extraction with zinc dibenzy! dithiocarbamate. Amer. J. Clin. Purhol. 41, 22-26 (1964). 4. Matsuba, Y., and Takshashi, Y., Spectrophotometric determination of copper with N,N,Nl,Nl-tetraethylthiuram disulfide and an application of this method for studies on subcellular distribution of copper in rat brain. Anal. Biochem. 36, 182-191 (1970). 5. Mikat-Devic, M., A sensitive method for determination of serum copper. Clin. Chim. Acta 7, 788-793 (1962). 6. Peterson, R. E., and Bollier, M. E., Spectrophotometric determination of Anal. Chem. 27, serum copper with biscyclohexanoneoxalyldihydrazone. 119.5-1197 (1955). 7. Planas, J., and Balasch, J., Correlation between serum iron and copper in different animals. Rev. Espan. Fisiol. 26, 91-94 (1970). 8. Reiff, B., and Schnieden, H., Plasma copper and iron levels and plasma paraphenylore diamine oxidase activity (plasma copper oxidase activity) in kwashiorkor. Blood 14, 967-971 (1959). 9. Rice, E. W., Spectrophotometric determination of serum copper with oxalyldihydrazide. J. Lab. Clin. Med. 55, 325-328 (1960). 10. Smith, G. F., and McCurdy, W. H., Jr., 2,9-Dimethyl-1, lo-phenanthroline new specific in spectrophotometric determination of copper. Anal. Chem. 24, 371-373 (1952). II. Smith, G. F., and Wilkins, D. H., New calorimetric reagent specific for copper. Anal. Chem. 25, 510-511 (1953). 1.2. Stark, G. R., and Dawson, C. R., Spectrophotometric microdetermination of copper in copper oxidases using oxalyldihydrazide. Anal. Chem. 30, 191194, 1958. 13. Watkins, R., Weiner, L. M., and Zak, B., Determination of copper, iron, and zinc from a single small sample, Microchem. J. 16, 14 (1971). 14. Welshman, S. G., The determination of serum copper, Clin. Chim. Actn 5, 497-498 (1960). 15. Worwood, M., and Taylor, D. M., Subcellular distribution of copper in rat liver after biliary obstruction. Biochem. Med. 3, 105-I 16 (1969).