Direct Measurement of Zinc in Plasma by Atomic ... - CiteSeerX

6 downloads 0 Views 957KB Size Report
CHEM. 25/8, 1487-1491 (1979). CLINICAL CHEMISTRY, Vol. 25, No. 8, 1979 1487. Direct Measurement of Zinc in Plasma by Atomic Absorption. Spectroscopy.
CLIN. CHEM. 25/8, 1487-1491 (1979)

Direct Measurement of Zinc in Plasma by Atomic Absorption Spectroscopy Submitters:

Evaluators:

J. C. Smith, Jr.,* Trace Element Research Laboratory, Veterans Administration Hospital, Washington, DC 20422 G. P. Butrimovitz** and W. C. Purdy,t Department of Chemistry, University of Maryland, College Park, MD 20741 R. L. Boeckx, Children’s Hospital, National Medical Center, Washington, DC 20010 R. Chu, Chief Chemist, Nutrition Laboratory, Veterans Administration Hospital, Albany, NY 12209 M. E. McIntosh, K.-D. Lee, J. K. Lynn, and E. C. Dinovo, Laboratory Service, Veterans Administration Hospital, Sepulveda, CA 91343 A. S. Prasad, Professor of Medicine, Department of Medicine, Wayne State University, Detroit, MI 48207 H. Spencer, Chief, Metabolic Section, Veterans Administration Hospital,

Hines, IL 60141 Present

addresses:

Nutrition Institute, U.S. Department of Agriculture, Science and Education Administration, Beltsville, MD 20705 ** Clinical Chemistry Division, University of Seattle, Seattle, WA 98195 t Department of Chemistry, McGill University, Montreal, Canada H3A2K6 *

Introduction The techniques now used to determine zinc in serum or plasma include colorimetry, neutron activation analysis, polarography, x-ray fluorescence, emission spectrography, fluorometry, and atomic absorption spectros#{231}opy (AAS) (1, 2). AAS techniques are preferred in the clinical laboratory because of their specificity, sensitivity, precision, simplicity, and relatively low cost per analysis. Before AAS burner heads capable of aspirating high-solid matrixes became available, preparation of plasma samples had to include precipitation and extraction. These pre-treatment techniques were time consuming, and a source of inaccuracies (3, 4). Therefore, a direct method is preferred if the accuracy of the method can be established. The greatest source of inaccuracy for flame methods, however, appears to be viscosity, with the differences it causes in aspiration rate between sample and working standard (5). In 1965, Sprague and Slavin (6) suggested a direct AAS method for serum zinc, the serum being diluted with an equal volume of water. The accuracy, as reflected by analytical re-

covery studies, was poor, and the burner head had to be modified to prevent clogging. In addition, the working standards were prepared in water and were not adjusted for differences in viscosity between sample and standard. In 1969, Hackley et al. (7) described an AAS method for zinc in plasma that was similarly diluted with de-ionized water. They prepared the standards in a dextran solution (30 gIL) to match the viscosities of the diluted plasma and eliminate the major error caused by the use of standards prepared in water. They concluded that “to avoid analytic error, it is necessary for the reference standard and the sample to have a similar viscosity.” In addition, Reinhold et al. (8) illustrated that the rate at which the sample flowed into the instrument was affected by the size of the capillary tubing used for aspiration, and suggested that differences in viscosity between the diluted serum and standards were responsible. They reported that at least a threefold dilution of serum with water was necessary for accuracy. To minimize errors from clogging, ion interferences, and differences in viscosity between sample and standard, others suggested dilutions as great as 20-fold (9-11). Such large dilutions weakened the signal, so that enhancing agents such as nitric acid or n-butanol were necessary. In addition to a relatively low signal-to-noise ratio, very dilute samples may also decrease precision because of greater pipetting errors. The optimum dilution is the one that does not clog the capillary and burner head but still yields an adequate signal. It was suggested (12) that serum diluted fivefold (one part plasma + four parts water) could be compensated for by use of zinc standards in glycerol/water (5/95 by vol). Accuracy has not heretofore been established for the use of working standards in glycerol with a direct-dilution method for plasma zinc. Therefore, we tested a one-step dilution of one part by volume of plasma with four parts of de-ionized water (13). The viscosity of the resulting solution was effectively matched by working standards that were prepared in glycerol/water (5/95 by vol).

Reagents and Materials1 Glycerol

solution.

Dilute

50 mL of glycerol

to 1000 mL with

de-ionized water.2 Our supplier of glycerol, certified ACS (99.4%), was Fisher Scientific Co., Silver Spring, MD 20910. Glassware. All volumetric glassware must meet NBS Class i All reagents, diluents, and containers used were regarded as possible sources of zinc contamination and were subjected to a continuous quality control program. Although sources of materials are given, other sources may prove satisfactory. 2 De-ionized water with a specific resistance of at least io ohms

at 25

#{176}C. CLINICAL CHEMISTRY, Vol. 25, No. 8, 1979

1487

A specifications. Glassware and Pasteur pipettes are acid washed, soaked in disodium ethylenediaminetetraacetate (EDTA) solution (10 g/L) for 24 h and rinsed six times with de-ionized water. Our acid-washing mixture was “No-Chromix” (Godax Laboratories, New York, NY 10013); disodium EDTA was from Fisher Scientific Co. Serological

pipettes

and tubes.

The disposable

serological

pipettes3 and polystyrene tubes4 we used in this study contributed no detectable zinc to the sample and therefore required no pretreatment. However, one cannot simply take this for granted, and pipettes and tubes should be checked regularly. Para film.

Parafilm

(a type of paraffin wax/elastomer squares. One source is Fisher

sheeting) is cut into 2.5-cm Scientific

Co.

Standards (a) Stock standards.

Primary

standard,

1000mg

of zinc per

liter: Dilute 10 mL of nitric acid to 50 mL, dissolve 1.000 g of zinc metal in this and further dilute to 100 mL. We used “Ultrex” nitric acid from J. T. Baker Chemical Co., Phillipsburg, NJ 08865; our zinc metal was zinc powder, 200 mesh, from Alfa Inorganics, Beverly, MA 01915. (b) Working standards, 100,200,300, and 400 tg of zinc per liter. Deliver 1 mL of 1000 mg/L zinc standard into a 100-mL volumetric flask and dilute to volume with glycerol/water solution (5/95 by vol). Mix by inverting at least 16 times. Place aliquots of this intermediate stock (1,2,3, and 4 mL) into four 100-mL volumetric flasks and dilute to volume with the glycerol/water mixture. The standards (0.1, 0.2, 0.3, and 0.4 mg of zinc per liter) correspond to apparent plasma zinc concentrations of 500, 1000, 1500, and 2000 g of zinc per liter. Prepare a working curve daily from fresh standards. Calculate the concentration of zinc in the plasma directly from the curve. (c) Pooled plasma for quality control. Pooled plasma can serve as a reference to monitor inter-day reproducibility.6 Plasma is ideally obtained as a large (200 mL) single spacimen from

a

normal healthy individual.

Control plasma should be

the plasma from the cells. Transfer the plasma into polyeth-

ylene storage vials, taking care to buffy coat or packed cells because relatively high amounts of cellular this reason, discard all hemolyzed stored at -20 #{176}C.

prevent disruption of the these cells can introduce zinc into the plasma. For samples. The samples are

Procedure Allow plasma samples to come to room temperature

and

then mix each sample by gently inverting the tube six times. working standards as previously described. Deliver 0.5 mL of plasma sample with a serological pipette into a 16-mm plastic test tube. Add 2.0 mL of de-ionized water and immediately mix the solution for 30 s. Repeat for plasma samples in groups of 10. Similarly prepare a control sample of pooled plasma. Establish instrumental and gas-flow settings and aspiration rate precisely, to optimize signal and minimize background noise. The instrumental settings shown in Table 1 apply to the instrument we used in this study. Once the aspiration rate is optimized with 10-mL aliquots of water, lock the nebulizer flow adjustment in place.’2 Aspirate glycerol/water solution (5/95 by vol) into the luminescent flame and set the baseline to read 0.000 ±0.001 absorbance (A). Take a baseline reading before and after each sample and reset the baseline as required. Sample the zinc working standards sequentially from most dilute to most concentrated, aspirating until the reading is stable (±0.001 absorbance); then record six successive 1-s integration readings. Average the readings for each sample; the resulting values are used to establish the working curve, preferably by use of a regression least-squares fit. Mix again and aspirate the standardized pooled plasma sample. Calculate the concentration from absorbance readings by interpolation from the working curve, as illustrated in Figure 1. Results must be within 20 g/L (ca. 3%) of the mean previously established.”4 Prepare

Results

negative for hepatitis B antigen and free of hemolysis. Store Curves in 1-mL portions in individual polyethylene vials7 at -20 #{176}C. Standard Working Thaw an aliquot at room temperature and analyze it with the Figure lisa typical daily working curve for zinc standards plasma samples. prepared in the aqueous glycerol. Known zinc concentrations of plasma samples followed the working curve closely. In Blood Collection and Preparation of Samples contrast, curves from the zinc standards prepared in solutions Collect 3 to 5 mL of whole blood by venipuncture, using with no or 100 mL of glycerol per liter differed significantly all-plastic polyethylene syringes8 and stainless-steel needles9 (p