Microdeterm inalion of Serum Copper Ruby M. Summers, Psychiatric and Psychosomatic Research Laboratory, V. A. Hospital, Houston, Tex.
T
possibility that serum copper levels may have a role in psychotic diseases ( 1 , 2, 4 ) made it desirable to develop a method that could be adapted to automatic analytical procedures. Of the several basic procedures examined] the oxalyldihydrazide-acetaldehyde method of Stark and Damon (7) appeared to be the most amenable t o this adaptation. It was necessary to remove the proteins, since they interfered with the determination. The sensitivity of the method was increased materially, so that the low concentration of copper found in serum could be measured more accurately. Precise and rapid determination of serum copper in large numbers of samples is feasible by means of the automatic method presented here. Furthermore, only general supervision by a skilled technician is required. The pressurized system used represents the first application of automatic procedures to niicrodeterminations. HE
METHODS
Modified Manual Procedure. Serum (2.0 ml.) and 1.0 ml. of 1N IlCl are mixed in a glass-stoppered centrifuge tube. After the mixture is heated for 10 minutes in a simmering water bath, i t is cooled t o room temperature and 1.0 ml. of 10% trichloroacetic acid is added with stirring t o precipitate the protein. Fifteen minutes later the sample is centrifuged until the supernatant liquid is clear (approximately 10 minutes). A 2.5mi. aliquot of the supernatant liquid and 0.5 nil. of the freshly prepared oxalyldihydrazide-acetaldehyde color reagent are mixed in a 10-mni. cuvette.
After 20 minutes, the sample is read in a spectrophotometer against a reagent blank a t 550 mp. The color reagent consists of 10 ml. of 0.3y0 oxalyldihydrazide, 2.5 ml. of concentrated ammonium hydroxide, and 0.5 ml. of acetaldehyde. Automatic Analytical Procedure (6). Instead of precipitation of the protein as above, a dialysis unit is employed. It is necessary to pressurize the system to force sufficient copper through the dialyzer for accurate measurement. A schematic flow diagram of the system is presented in Figure 1. The pressurization led t o several problems in maintaining the constant pressure that is mandatory. First, with the equipment available at present (AutoAnalyzer, Technicon Instruments Corp., Chauncey, N. Y.), it was necessary to increase the tension on the platen of the pickup side of the pump. Next, the system must be in continuous operation and must contain the fluid-air segments up t o and through the dialyzer before the first standard or sample is aspirated. The same applies to the last 6 to 8 blanks (0.9% sample-Le., KaC1) are required a t the start and finish of a run. Standards (diluted with 0.9% NaC1) separated by cups of saline are inserted a t the beginning and end of a run. Before the first unknown serum is aspirated] the four preceding cups should contain alternate serum
and saline blanks. Thus, the pressure relationship can be maintained satisfactorily, and this fact is utilized to achieve differentiation between samples on the recording-Le., the unknown serum samples are interspersed with saline blanks in the turntable. Known serum samples may be placed in the turntable a t will to check on accuracy. The turntable is operated a t the rate of 20 changes per hour. Slightly more than 1.2 ml. of serum are required for each determination. After the samples are aspirated, air and 12v " 2 1 (to liberate the copper from the protein) are introduced. The serum passes through the water bath (37" C. for 3 minutes) and the dialyzer using a D-30 membrane (Technicon); a t this point pressure is applied. The dialyzate is picked up in a stream of 0.9% saline that already contains air bubbles to prevent mixing of samples, and the color reagent is added to this stream. A 9-minute delay coil permits maximum color development in the solution. It then passes through a 10-mm. continuous-flow cell of the colorimeter (550 mp), and the absorbance is recorded automatically. The color reagent is prepared daily by mixing 300 ml. of 0.3% oxalyldihydraaide, 40 ml. of concentrated ammonium hydroxide, and 4 ml. of acetaldehyde. This yields sufficient reagent for I1 to 12 hours of continuous flow of the systerr, (analyBis of more than 100 samples).
Figure 1. Component parts for pressurized system for automatic analysis of serum copper Air 2. 1.ON HCI 3. Sample pickup 4. 0.9% NaCl 5. Air 6. Color reagent 7. Pressure line 8. Water bath containing dialyzer 1.
Mi./Min. 0.60 0.42 1.20 0.42 0.32 0.32 1.20
Tubing, inch 0.040 0.035 0.056 0.035 0.030 0.030 0.056
oJ
, 50
100
150
200
250
A G . Copper/100 ml.
Figure 2 . Sensitivity of copper determination obtained with various methods VOL. 32,
NO. 13, DECEMBER 1 9 6 0
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Comparison of Methods for Determination of Copper in Serum (pg. per 100 ml.) QxaiyldihydrazideAutomatic Analysis Pooled Serum Acetaldehyde Batho~uproine~ Cuprhoneb Nonfasting Sample 1 217 167 157 Mean 168 9.3 3td. dev. 8. I 8.5 1.5 217 Sample 2 183 173 188 Fasting 129 N.U.d 1224 122 Sample 30 1.4 Std. dev. Sample 4O 134 142 125 323e 2 .3 Std. dev. t a b l e I.
0
volved, and therefore increases the number of samples that can be analyzed by a technician daily. ACKNOWLEDGMENT
The author is indebted to Roy B. Mefferd, Jr., of this laboratory for his suggestions and to Gerald Kessler of Technicon Instruments Corp. for suggesting the pressurized dialysis system. LITERATURE CITED
2,9-Dimethyl-4,7-diphenyl-l,lO-phenanthroline.
Biscyclohexanone oxalyldihydrazone. Single determinations. d Not determined. 8 Triplicate determinations. b
c
RESULTS
The oxalyldihydrazide-acetaldehyde procedure was the most sensitive of the three methods tested (Figure 2). The three manual methods (3, 5) and the automatic procedure gave comparable serum copper values (Table I), but there was a striking increase in precision
with the latter procedure (standard deviation of 1.5 us. values over 8 with nianual procedures). Besides being less precise, the manual procedures are quite tedious and timeconsuming. The pressurized automatic procedure not only increases precision, but also greatly reduces the labor in-
(1958). (5) Peterson, R. E., Bollier, M. E., ANAL. CHEM.27, 1195 (1955). (6) 8keggs. L. T.,Jr., Am. J. Clin. Pathol. 28,31i (1957). (7) Stark, G. R.,Dawson, C. R., ANAL. CHEM. 30,191 (1958).
New Teehniqwe for Rapid Determination of Resonance Positions of NMR Spectra
B, H. Arison and N. R. Trenner, Merck Sharp h Bohme Research Laboratories, Division with experience in NMR spectroscopy will appreciate the need for a technique more convenient than interpolation for determining resonance positions. One can avoid this somewhat tedious procedure by resorting to a variable scale such as developed by the Gerber Scientific Go., 162 State St., Hartford, Conn. An alternative approach is reported which uses a Sirnmon Model D3 Automega enlarger with a 135-mm. lens and a suitably engraved scale on a 4 X 5 inch glass plate (Figure 1). The scale image is simply projected onto the spectrum, adjusted to the proper size by lining up with an appropriate reference and side band(s), and the resonance positions are read directly. With this HOSE
Table i. Cornparison of Results Obtained by Scale and Interpolation Methods Scale Interpolation 4.81 6.12 6.25 7.50 7.91 8.77 9.09 9.19
4.80 6.12 6.25 7.48 7.96
8.77
9.08 9.18
technique, the reading time for most spectra can be reduced to approximately 1 minute. The particular scale designed by the authors is in shielding numbers [Tiers, 6. 71. D., J . Phys, Chem. 62, 1151 (195S)l (any other scale, of course, can be engraved) with lines spaced a t 0.05-7 intervals and heavier lines every 0.5 r. One can readily interpolate to the nearest 0.01 T (0.6 cycle at 60 &,IC.),a n accuracy more than adequate for general use. Table I illustrates the agreement
of Merck & Co., Inc., Rahway, N. J.
between the two methods from a spectrum chosen at random. The illumination of the scale is sufficient for use in an ordinary lighted room. A range of 7.5 units is adequate since it covers the bulk of proton resonances. ACKNOWLEDGMENT
The scale was prepared with the help of Fred Drexel of the Scientific Glass Apparatus Go., Bloomfield, N. J.
