BIOCHEMICAL MEDICINL Id, 378-383 t 1975)

Application of Selective Demasking to the Spectrophotometric Assay of Erythrocyte Zinc

PAUL. CARTER

Previous reports have confirmed the presence of elevated concentra- tions of red cell zinc in patients with chronic lymphocytic leukemia. and decreased levels have been reported in patients with Hodgkin’s disease as well as cirrhosis ( 1.2). Recent studies suggest the presence of erythrocyte zinc deficiency in sickle cell anemia (3). In an effort to monitor red cell zinc activity simply. a rapid. specific spectrophotometric determination has been developed, evolved from a recent communication from this labora- tory in which human serum was assayed for zinc using a methodology of selective demasking which exploited the reaction of a sensitive zinc- combining ligand, 1,2-(pyridylazo)-2-naphthol (PAN). in a buffered protein-free milieu containing a solubilizing detergent. obviating the need either for digestion of the sample or isolation of the complex from the reaction mixture (4).

MATERIALS AND METHODS

H~nzarz hloocl. Early morning specimens were obtained from fasting individuals, essentially laboratory personnel, 23 to 45 yr of age. having had no previous exposure to drugs for a period of at least 1 week nor presently amenable to any known acute or chronic disease. Suitable precautions were implemented to avoid contamination during blood collections by obtaining a freshly shed specimen in a Monoject disposable syringe rinsed with heparin and immediately transferred to a chemically clean container. An acceptable alternative was to permit the blood to partially fill a green- stoppered Vacutainer without touching at any time the sealed rubber closure, a source of zinc contamination.

Rragrnfs. Protocol for the preparation of reagents is similar to the previously described assay for serum zinc (4) with one exception. A prorcitz

hcmol~sate-prc~cipitating solution, 2.4%, ascorbic acid and 0.24 N HCI in 1 I .3% trichloroacetric acid (TCA), is prepared by transferring 5.0 ml of the stock TCA to a 50-ml volumetric flask containing I .2 g of ascorbic acid and 1 ml of 12 N HCI and diluting to volume. All aqueous solutions were

37x

SPECTROPHOTOMETRIC ASSAY OF RED CELL ZINC 379

prepared from water purified by distillation. Reactions were performed with disposable polyethylene graduated pipets, encompassing a volume range between 0.10 and 1.00 ml. By using polyethylene test tubes simul- taneously as reaction vessels and absorption cuvettes having a 12-mm light path adapted to the Coleman Junior spectrophotometer, severity in the contamination error was minimized.

Zinc determination. A suitable aliquot of centrifuged blood cells sepa- rated from supernatant plasma was washed three times with isotonic saline before hemolysis with a fivefold volume of metal-free distilled water. This solution, spun free of its stroma, was used for the analysis of zinc and hemoglobin. To 500 ~1 of prepared hemolysate was added 1 .O ml of protein hemolysate-precipitating solution. The test tube was capped to prevent evaporation and allowed to stand for 5 min after mixing. After centrifuga- tion, 1.0 ml of the clear centrifugate was added to 1.0 ml of Tris buffer followed by 0.1 ml of complexing agent. After mixing, 0.1 ml of color reagent was added, and absorbance in the reaction vessel was read against water at 550 nm. Addition of 0.1 ml of demasking reagent, followed by mixing, liberated the zinc to form a complex which was read again at the same wavelength. Substitution of an appropriate blank and standard throughout the entire procedure is required for calculation of the total erythrocyte zinc concentration. The Ferrozine iron technique was used for the determination of hemoglobin (5).

Standards. ZnO was dissolved in concentrated HN4, and diluted to volume with water to yield a concentration of 1000 ppm. Working standards containing 50, 100, and 200 pg of ZnIdl were prepared by appro- priate dilutions from the stock. Plastic vessels which contributed minimal contamination were used wherever possible for storage. Reagent standards of the highest purity available but not further treated to eliminate contami- nation were used throughout the study.

Calculations. Micrograms of Zn/gram of hemoglobin in the hemolysate can be obtained from the hemoglobin concentration (grams/deciliter) and zinc concentration (micrograms/deciliter) expressed as the net spec- trophotometric absorbances bcifore and after demasking of the unknown, standard, and blank which obey Beer’s Law.

RESULTS

The relative molar concentration ratio of iron, copper, and zinc within the erythrocyte is about 100: 1: 1. By selective demasking prior to the PAN reaction, zinc can be assayed in the presence of an eightfold molar concen- tration excess of iron and copper without appreciable analytical error. Centrifugates from five hemolysate pools containing 35 to 50 mg of hemoglobin/ml, analyzed for iron and copper (6,7), yielded mean values of 50 to 90 pgldl, respectively, always less than the concentration of zinc

380 PAUL CARTER

present and well within the limits of acceptable analytical tolerance. It is concluded that iron-bound heme remains essentially unattacked within the precipitate.

Spectrophotometric titration of reaction mixtures with increasing con- centration of reducing agent indicated that stabilized metal complexing in the presence of cupro(1) and ferro(II) cyanide exists at a concentration of 6.9 mg of ascorbic acid/ml, about twice the requirement for protein-free serum centrifugates. hence the necessity for modifying the precipitating agent described.

The presence of electrolytes in an approximately fivefold excess concen- tration yielded a mean positive error of less than 2.0% when duplicate hemolysate pools of 5.0-ml volume, inoculated with IOO-ml aliquots of an aqueous mixture, containing 31.8 mg of NaCI. 161 mg of KCI. 24 mg of KH,PO,, 6.5 mg of CaCl,*2H,O, and 3.0 mg of MgCl, .6H,O in a total volume of 1 ml, were incubated at 37°C for several hours and analyzed for erythrocyte zinc by the proposed method. No appreciable difference was found in concentration of red cell zinc at different levels of packed cell mass nor was there more than 2% variation found in concentrates before and after washing, so that it must be assumed that trapped plasma concentra- tion must be at the minimal level of analytical tolerance. A viable alterna- tive for the selection of a suitable hemolysate might be to select an aliquot of cells from a well-packed centrifuged specimen at midlayer and hemolyze without further precautions.

Replicate introduction of micro-aliquots of ionic zinc in hemolysate pools spanning a threefold variation in zinc concentration resulted in a mean recovery of 96% as illustrated in Table I. The addition of 1 .O N HCI for 5 min in a boiling-water bath prior to protein precipitation in a reducing milieu did not perceptibly increase metal extraction efficiency. Analysis of

TABLE I

RECOVERY OF ZINC FROM L.\sto Rtr, C‘t L I 4

Hemolysate

zinc present

(NJ) ___-.

0.403

0.403

0.403

0.403 0.403

Ionic zinc Total zinc

added present Found Rccu\rrL

(a) (I*ia (P) !’ ; ~- ___~~__... - ~~ -. -~~~~ ~~~~ _ ~. ~~ _..

0.182 0.585 0.M' 101

0.364 0.767 0.730 95

0.546 0.949 0.893 93

0.728 1.131 I.070 95 0.9.10 1.313 1.267 9:

Mean 96

SPECTROPHOTOMETRIC ASSAY OF RED CELL ZINC 381

160- Estlmatlny equation

y 1108X~17 .

170- " = 14

r = 09533 .

160- syx = -173 .

7 P; 1462 - 0.

.

2 g l50-

7 2 1475

8

i: 130- .

. 120

I, I I I I I I 0 120 130 140 150 160 170 180

AmmIc Absorpt~onpy Zn/d\ iv.1

FIG. 1. Comparison of protein-free red cell hemolysate zinc concentration obtained by Zn-PAN complex after selective demasking and atomic absorption.

the packed precipitate obtained from the reaction of 10 ml of hemolysate with 20 ml of protein-precipitating reagent after appropriate washing and wet ashing with nitric acid and hydrogen ,peroxide was performed by atomic absorption. An error of less than 2% confirmed that zinc is effec- tively extracted from its protein source, and it appears that the metal is not in any way occluded within the interstices of the precipitate.

Comparison nith Other Twhniquc)s

Correlative determination of hemoglobin-free centrifugates by atomic absorption spectrometry, where reagent blank, standards, and specimens are aspirated from an identical matrix, was performed on a dual channel Model 810 Jarrel-Ash instrument at 213.9 nm.

The absolute variation from the reference atomic absorption measure- ment (8) on 14 specimens was 2.84%, and the arithmetical error was 1.11%. Analysis of 10 specimens from a healthy hospital employee population produced a mean ratio of 36.9 pg of Zn/g of hemoglobin. Normal values are identical with those found by emission spectroscopy, in which specimens were dry ashed at 450°C in a muffle furnace prior to analysis (9). Normal values obtained by an atomic absorption technique in which hemolysates are decomposed in nitric acid (10) as well as a spectrophotometric tech- nique in which Zincon is employed (11) are about 10% higher than values found by the procedure described here (Table 2). Analytical error is minimized by using a mass ratio as the expression of zinc concentration within the cell since a knowledge of only two variables is necessary: hemoglobin and zinc concentration within a specified hemolysate. The use

382 PAUL CARTER

Zak (4 (il. ( 1 1)

Prasad r’/ (il. ( IO)

Valberg v/ ol. (9)

This communication

39.6 i- 6.X 41.7 2 6.5 37.1 i- 6.5 36.9 t 2.4

Zincon by spectrophotometry

Wet ashing. atomic absorption

Dry ashing. emisbon \pectroxop)

PAN by spectrophotometr)

of other expressions, such as micrograms of Zn/volume of red cells (mil- liliters), micromoles of %/red cell, requires the additional task of recon- stitution of suspended cells after sedimentation as well as measurement of hematocrit and red cell count.

Erythrocyte zinc has been determined by neutron activation analysis (12) after wet ashing prior to a 6-hr reactor irradiation. Potentially it can be assayed directly by flameless atomic absorptions (13), provided a means can be found to mask the interfering iron. There are also spectrophotomet- ric techniques which require wet ashing of the protein background because of gross interference, but some workers have been unable to obtain quan- titative recovery by this route using perchloric acid and sulfuric acid in Pyrex glass vessels (14).

l’he advantage of the proposed assay described here lies in the perform- ance simplicity using unsophisticated instrumentation in a background matrix where the presence of excessive electrolyte and trace metal indige- nous to the red cell have only a trifling effect on the analytical accuracy. The 30-min stabilized Zn-PAN complex has a molar absorptivity coefficient of 5.28 x IO4 I mole-’ cm ‘. thus approaching the atomic absorption technique in sensitivity, and yieldingabout twice the sensitivity of the Zincon complex where hemolysate boiling prior to the formation of a I-min stabilized complex is required.

SUMMARY

Variation in red cell zinc concentration can be monitored simply through the application of selective demasking prior to the formation of a metal combining ligand. 1,2-(pyridylazo)-2-naphthol, (PAN) in a protein-free hemolysate. Favorable correlation obtained by the proposed method with dual channel atomic absorption spectrometry should make the method especially attractive where a limited choice of instrumentation is available.

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SPECTROPHOTOMETRIC ASSAY OF RED CELL ZINC 383

3. Oelschlegel, F. J., Brener, G. J., and Prasad, A. S.,Biochem. Biophys. Rcs. Commun. 53, 560 (1973).

4. Carter, P., Clin. Chim. Acta 52, 277 (1974). 5. Manasterski, A., Watkins, R., Baginski, E. S., and Zak, B., Z. Klin. Chum. K/in.

Biochrm. 11, 335 (1973). 6. Carter, P., Clin. Chim. Acta 39, 497 (1972). 7. Carter, P., Anal. Biochcm. 40, 4.50 (1971). 8. Rosner, F., and Gorhen, P.. J. Lab. C/in. Med. 72, 213 (1968). 9. Valberg, L. S., Holt, J. M., Paulson, E., and Szivek, J., J. Chin. Invest. 44, 379 (1965).

10. Prasad, A. S.. Shoomaker, E. B., Ortega, J., Brewer, G. J., Overleas, D., and Oelschlegel, F. J., C/in. Chem. 21, 582 (1975).

1 I. Zak, B., Nalbandian R. M., Williams. L. A., and Cohen, J., Clin. Chim. Acta 7, 634 (1962).

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13. Kurz, D., Roach, J., and Eyring, E. J., Anal. Biochum. 53, ,586 (1973). 14. Berl’enstram, R., Acta Paediat. (Uppsala) 41 Suppl. 87 (1952).