Comparative measurements of zinc-70 enrichment in human plasma samples with neutron activation and mass spectrometry

163

Clinica C&mica Actu, 114 (1981) 163-171 Elsevier/North-Holland Biomedical Press

CCA

1819

Comparative measurements of zinc-70 enrichment in human plasma samples with neutron activation and mass spectrometry Morteza

Janghorbani

a-*, Vernon Lawrence

R. Young a, John W. Grarnlich A. Machlan b

b and

a Nuclear

Reactor Laboratory, Deparrment of Nutrition and Food Science, Clinical Research Center, Massachusetts Instirute of Technology, Cambridge, MA 02139 (U.S.A.) and b Center for Analytical Chemistry, National Bureau of Standardr, Washington, DC 20234 (U.S.A.) (Received

December

17 th, 1980)

Summary

Radiochemical neutron activation analysis (RNAA) is compared with isotope ratio mass spectrometry (IRMS) for the measurement of “Zn isotopic enrichment in human plasma following oral administration of the isotope. It is shown that both techniques are suitable for this purpose, although IRMS yields more precise data. Each method, with its advantages and limitations, can be realistically employed to study kinetics of appearance of 70Zn in plasma obtained during human metabolic studies.

Introduction

Plasma appearance curves for zinc following oral administration in humans have so far been studied with either radiozinc [l] or normal zinc [2]. The former method is restricted in regard to general use because of issues related to administration of radioisotopes, while the latter requires oral intakes of zinc significantly greater than the normal daily intake of the mineral. The stable isotope ‘OZn is naturally present at 0.62% atomic abundance, being the least abundant of all stable isotopes of zinc. Therefore, it could potentially provide a suitable vehicle for dietary enrichment and plasma appearance studies. Thus, it is of considerable interest and importance to consider whether or not oral administration of this isotope at physiologically relevant levels allows measurement of “Zn enrichment in human plasma to the extent that would permit investigation of plasma appearance kinetics of this isotope. Accurate measurement of ‘OZn enrichment in plasma under normal nutritional conditions is difficult due to two primary factors. First, in a typical human study involving time dependent measurements, the amount of plasma available at any * Correspondence to be addressed to: M. Janghorbani, Street, Cambridge, MA 02139, U.S.A.

ooO9-8981/8l/oooO-0000/$02.50

0 Elsevier/North-Holland

Nuclear

Reactor

Biomedical

Laboratory,

Press

MIT,

138 Albany

164

given time is restricted, often to a few milliliters. This sample size provides total 70Zn content of only a few nanograms. Secondly, the expected total plasma increase due to the administration of the isotope is at most only a few percent of the oral dose [ 11. Therefore, it is not readily apparent whether existing analytical methodology will allow such measurements to be carried out. We have explored this problem using both radiochemical neutron activation analysis (RNAA) and isotope ratio mass spectrometry. (IRMS) and discuss our comparative results in this manuscript. Materials and methods Human experiments Following an overnight fast, four healthy adult male volunteers, ages 24- 37 years and weighing 54-67 kg, consumed a single glass of orange juice containing 3.2 mg 70Zn (Oak Ridge National Laboratories, Oak Ridge, TN) at 9:00 a.m. Venous blood samples were withdrawn from the antecubital vein every 15 ruin for the first two hours, at half-hour intervals for the next 1.5 h, and then hourly until 8 h post isotope ingestion. Two separate blood samples were also withdrawn from each subject immediately prior to isotope administration. The subjects consumed a light lunch consisting of ham sandwich, canned fruit, and milk or coffee. Blood samples were withdrawn into Zn-free syringes containing no anticoagulant, and after clot formation the serum was separated. The separated samples were stored in a deep freezer until used for analysis. RNAA measurements The procedure followed to measure 68Zn and 70Zn with RNAA has been described in detail elsewhere and its precision capabilities and limitations have been discussed 131.

TABLE

I

DATA COMPARING RICHED SAMPLES

PRECISION

OF IRMS

AND

RNAA

FOR

‘cZr#‘sZn

RATIOS

IN UNEN-

I SD.

Method

Ratio *

Average?

IRMS

0.03254 0.03244 0.03244 0.03249 0.03246

0.03247 I- O.oooo4 (0.13%)

RNAA

0.~~3 0.00838 0.00858 0.00784 0.00852 0.00708

0.~824~0.~8

(8.3%)

* For IRMS data samples consist of either high-purity zinc wire or unenriched reported as relative atomic abundance ratios for 70Zn/6RZn.

human

plasma.

Data are

For RNAA data samples consist of unenriched plasma samples from four different individuals. Data are given as ratios of peak areas for “*Zn (386 keV)/69mZn (439 keV) corrected for radioactive decay.

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The ratio of “Zn to 68Zn in unenriched plasma samples has been determined for six samples of human plasma with a C.V. of 8% (see Table I). Therefore, the method is capable of performing these measurements in unenriched plasma samples to within 8%. The basic factor determining the measured precision is related to the counting statistics of the 386 keV (70Zn, ‘lmZn [386 keV]) so that for “Zn enriched samples the precision will be better depending on the degree of enrichment [3]. IRMS

measurements The mass spectrometric technique utilized in this study is based on IRMS with thermal ionization. The methodology and instrumentation has been described before [4,5]. The procedure as used for this work is described below. The serum in each polyethylene vial (1- 2 ml) received was transferred to Teflon beakers and decomposed by heating after the addition of 3 g of HNO, and 5 g of HCl. The dissolved samples were loaded onto ion exchange columns prepared in the following manner: A 5-ml plastic syringe with a porous plastic disk placed in the bottom was filled to the 5-ml mark with AG l-x8, 100-200 mesh resin. The column was cleaned by adding 20 g of 4 mol/l HNO,, 26 g of H,O, 20 g of 8 mol/l HCl and 20 g of H,O. Approximately 5 g of 1.5 mol/l HCl was added to the column to condition it. The sample beaker was rinsed with 5 g of 1.5 mol/l HCl. Other elements were eluted with 20 g of a solution of 1.5 mol/l HCl and 1 mol/l HF followed by 25 g of 0.5 mol/l HCl and the zinc was eluted from the column with 30 g of 0.02 mol/l HCl. The zinc containing fraction was evaporated to dryness, a few drops of HNO, were added and the solution was heated to decompose any organic residue. The samples, in Teflon beakers, were evaporated to dryness and transferred to the mass spectrometry laboratory. The samples were analyzed by solid sample thermal ionization mass spectrometry using the silica gel-phosphoric acid technique for ionization enhancement [5,6]. Before sample loading, the rhenium filaments were degassed in a vacuum and under a potential field for 30 min at approximately 2000°C. A lo-p1 drop of a colloidal silica gel solution was placed on the filament and dried with an electrical current of 1 A through the filament for 5 min. This step was repeated with a second drop of silica gel solution. The zinc sample was dissolved with 3 drops of 0.2 mol/l HNO,. One drop of this solution was placed on the silica gel layer and dried at 1 A for 5 min. A lo-p1 drop of 0.75 nmol/l H,PO, was then added to the filament and dried at 1.3 A for 5 min and 1.5 A for an additional 5 min. During the above steps, a heat lamp placed above the filament aided the drying. The temperature at the filament resulting from the heat lamp was 50°C. After the phosphoric acid drying steps, the heat lamp was turned off and the filament current was slowly increased until visible fumes were present. After fuming off the excess H,PO,, the filament was momentarily (1 s) increased to a temperature of about 900°C. The filament was then immediately loaded into the mass spectrometer. The mass spectrometer analyses were performed according to the following procedure. The filament was heated initially to a temperature of 1500°C. After 5 min the temperature was increased to 1600°C. This produced a total zinc signal intensity of approximately 6 X 10 -‘I A which decayed continually during the analysis. The 70Zn/68Zn ratios were measured during the time period of 30-50 min into the analysis. Because of a small electron scatter peak in the vicinity of mass 70, which continually decreased in magnitude, baseline measurements were made every 5 min

166

and data collected between baseline measurements was computer corrected to the average before and after baseline measurements. Results and discussion

Measurement precision of the two methods IRMS is expected to be capable of much greater measurement precision than is RNAA. Data related to the measured precision of these two methods are given in Table I for unenriched samples. The data are consistent with the expected precisions of the two techniques [3,5]. It is clear then that IRMS yields data with significantly better precision than RNAA. This is a major attribute of mass spectrometric technique to the present problem since it is expected that plasma enrichment of 70Zn following oral administration of physiologic doses of the isotope will be small [l]. Background contamination The isotope ratio measurements performed on unenriched samples are unaffected by any contamination arising from reagents. However, isotopic ratio measurements performed on enriched samples will be affected by contamination from reagents. Consequently, since the expected plasma enrichment is relatively small, the measured enrichment ratio may be smaller than the true value. Fortunately, this is of little consequence since the isotopic enrichment ratio is really not the final parameter of significance in plasma appearance studies. Instead, the parameter of interest is the fraction of the oral dose present in the plasma at any given time. To arrive at this value, the plasma content of 70Zn originating from any source other than the 70Zn given as the label is calculated from the measured value of 68Zn (or any other stable TABLE

II

COMPARATIVE Time (min)

ISOTOPE

post admin.

Unenriched 15 30 45 60 75 90 120 150 180 210 240 210 330 390 450

RATIO

DATA

FOR RNAA

AND IRMS

70Zn/68Zn)IRMS

(

0.00824 2 0.00068 0.00956 0.01001 0.0124 0.0145 0.0 162 0.0163 0.0164 0.0162 0.0144 0.0144 0.0172 0.0161

0.03247 k 0.00004 (0.13%) 0.03817 0.04934 0.05659 0.06193 0.06501 0.07067 0.07348 0.07339 0.06697 0.07120 0.07378 0.07085

3.94 3.99 4.93 4.56 4.27 4.01 4.33 4.48 4.53 4.65 4.94 4.29 4.40

0.0128 0.0131

0.05916 0.06007

4.62 4.59 av. 4.45 20.30

(70Zn/68Zn)IRMS * k=

(PA,,,/PA,,,)RNAA

k*

(PA),,,/(W,39

(6.7%)

167

y=O.2061

X + 0.001178

1.8 1.7 1.6 -

g -

l.3-

x : *

1.2I.1

2 a

I.O-

;:

0.9

-

-

CD : 0.8;I &

0.7

-

0.6

-

0.30.2

-

0.1

-

I

I

I

0.01

0.02

003

I

I

ATOMIC ABUNDANCE 7oZn/% MEASURED

Fig.

1. Correlation

I

OD4 CO5 006

of 70Zn/68Zn

I 0.07

I

I.

0.08 QOS 0.100

RATIO OF WITH IRMS

enrichments

in plasma

between

RNAA

and IRMS measurements.

isotope of zinc) and the natural isotopic ratio of “Zn to 68Zn. The difference between total plasma “Zn at any given time and this correction value is then expressed as the fraction of the administered dose. However, for the purposes of comparing the two methods of IRMS and RNAA using the measured enrichment ratio in different plasma samples from the present study, one must take into account the fact that the degree of background contamination in the two procedures was different. This then affects the measured values of “Zn/‘Zn on a given sample as obtained from the two different techniques. The estimated zinc contribution from the reagents during IRMS measurements is about 10 ng per 2-ml sample, the comparative figure for the present RNAA procedure is estimated at about 0.5 pg. It should be feasible to reduce the blank contribution in the RNAA to significantly below this figure, should this be deemed necessary. The consequences of the significantly different degree of blank correction in the two methods will result in the measured enrichment ratios to be lower for the RNAA than for IRMS (and the true value). However, as long as the inter-sample blank correction is constant the two techniques can still be compared. Since the level of “Zn originating from all unenriched sources is determined for each sample separately, the relatively high blank correction is not a limitation.

168

Comparison between RNAA and IRMS analyses The isotope ratio measurements performed on each of 15 plasma samples obtained from one subject (M.J.), as well as the average values of unenriched measurements (see Table I) are given in Table II for the two methods. The mass spectrometric values are given as the relative atomic abundance ratio of the two isotopes while the neutron activation results are in terms of gamma line peak area ratios for 7’mZn/69m Zn. This is unimportant for comparative evaluation of the two methods since the ratio of the RNAA and IRMS measurements on each sample should be a constant related to instrument parameters. This constant (k) which should be reproducible within the combined precision merit of the two methods, has also been calculated in the table for each sample. Consistent with our expectations based on the achievable precision of the RNAA method, the instrument parameter is constant to within 6.7% indicating that the individual variabilities are in fact due to the precision achieved with RNAA. The comparative isotope ratio data are also correlated in Fig. 1 and show a correlation coefficient ( r2) of 0.91. As seen from the data given in Fig. 1, the greatest absolute error between the two techniques is 11% of its value as determined by IRMS. Therefore, these data indicate that both RNAA and IRMS provide consistent measured values for the enrichment of “Zn in human plasma. However, mass spectrometric measurements are about fifty times more precise than can currently be achieved with RNAA. Practical aspects of RNAA and IRMS The foregoing data clearly show that both RNAA and IRMS yield consistent isotope ratio measurements for enrichment studies of “Zn in human plasma. The IRMS measurements can be carried out to a greater degree of precision which should permit accurate enrichment measurements at levels significantly lower than can be achieved with RNAA. However, the ultimate usefulness of each method is determined also by its measurement precision capability in relation to the expected enrichment of plasma samples. In addition, the eventual usefulness of a method is TABLE

III

KINETICS

OF APPEARANCE

Time post admin.

OF “Zn

PLASMA

% of dose in olasma

(h)

0.25 0.50 0.75 1.00 1.25 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.50 6.50 7.50 24.00 * Mean value for four subjects,

IN HUMAN

0.04~0.03 0.06 k 0.03 0.17~0.06 0.24kO.07 0.22CO.14 0.28kO.15 0.3OkO.12 0.27kO.11 0.28 t 0.09 0.27 k 0.08 0.37’0.15 0.30~0.10 0.17~0.05 0.15~0.04 0.18~0.07 0.10-c0.04

1 S.E.M.

*

169

determined by the practical consequences of its use in the solution of metabolically and nutritionally significant problems. Since a typical human metabolic study involving kinetic studies of plasma 70Zn appearance might require analysis of a few hundred plasma samples, each method must be evaluated, not only in terms of its ultimate precision capability-provided both yield precision useful for studies of interest-but whether it is practical. Here, we have set forth a brief discussion of practical aspects of these two methods of isotopic analysis as applied to the present problem of kinetic studies of 70Zn appearance in a medium size metabolic experiment which may yield about 200 samples for analysis. Relative precision

IRMS is superior to RNAA; but RNAA is satisfactory since the achieved plasma enrichment for 70Zn is large compared to the precision of the method. Present availability RNAA: Several research reactors are currently

available both in the U.S. and Europe with the neutron flux capability for this work. The principles and the necessary instrumentation are well established, given appropriate radiochemical expertise at the various sites. IRMS: Thermal ionization mass spectrometers capable of the accuracy and precision needed for this work are available at numerous university and government laboratories worldwide. Overall sample throughput RNAA: The necessary analyses for a typical 200-sample study can be completed

within a 60-day normal work schedule, assuming the reactor is available during the normal working hours (8 h/day) for 25 days during this time. ZRMS: Chemical separation and purification of zinc from 200 plasma samples would require approximately 40 days of normal work schedule for an experienced separations chemist. Samples could start being delivered to the mass spectrometry laboratory after the first week. Allowing time for the periodic analysis of standards and for routine instrument maintenance, approximately 90 days would be required to complete the mass spectrometric measurements. The advent of fully automated mass spectrometers would reduce time for analysis but the availability of this type of instrumentation is currently limited. Manpower requirements RNAA: For trained personnel, 60 days of one chemist and approximately

25 days of a technical assistant would be needed to complete analyses of 200 samples. IRMS: With experienced personnel, approximately 42 days for a chemist and 90 days for a mass spectroscopist would be required to complete the analyses. costs

Both methods require significant capital outlay and experienced personnel. The RNAA procedure can be carried out at the existing reactor facilities throughout the world. The mass spectrometry expertise could be acquired for individual laboratories, but the capital outlay will be significant.

170

Precision requirements

Since the two methods are capable of such widely different measurement precision, it is necessary to evaluate their capabilities in relation to measurement precision requirements of a given study. Thus, for example, Aamodt et al. fl] have investigated plasma appearance kinetics of radiozinc and have shown that plasma appearance of radiozinc corresponds to a peak value of 1.3% of the ingested dose. Furthermore, after 5 days post ingestion, plasma radiozinc had been reduced to 0.5% of the intake level. If in a typical metabolic study, a single 3-mg dose of 70Zn is ad~nistered whose kinetic plasma appearance is assumed similar to the data of Aamodt et al., then at peak appearance the expected 70Zn plasma enrichment should be about 200% of the plasma “Zn baseline level (an increase in 70Zn enrichment ratio from 0.62% to 1.83%), at 5 days post administration the corresponding value being 75% (0.62% vs. 1.09%). Under these conditions, RNAA should be satisfactory with its attainable precision of 5- 10%. We have explored this issue, using RNAA as the analytical measurement method, in four subjects who consumed a single glass of orange juice containing 3.2 mg “Zn after an overnight fast. The results of “Zn plasma appearance kinetics expressed as percent of oral dose are given in Table III. The data clearly show that under these conditions plasma appearance of 70Zn can certainly be measured, at least over the 24-h post ingestion period. Furthermore, the inter-individual variabilities measured in this experiment are very similar to those reported by Aamodt et al. [l], indicating that the measurement precision of RNAA does not appear to be an overriding determinant in these results. The 7’mZn/69mZn peak areas for the 24-h post ingestion plasma sample for the four subjects corresponds to 0.~908 t 0.00037 (1 S.E.M.) as compared with the corresponding value for the unenriched samples of 0.00824 -+ 0.00028 (1 S.E.M.). It should be pointed out here that the extent of plasma peak appearance measured by us in the present study is less than reported by Aamodt et al. [I]; 0.3% of dose compared with 1.3% in the latter study. Hence, where the extent of “Zn appearance in plasma resembles that of Aamodt et al. [I], the “Zn/‘*Zn would be about four times greater than that found in the present study. Therefore, it appears that RNAA provides satisfactory precision of measurement for these studies. Conclusions

The results from this comparative study indicate that both RNAA and IRMS yield consistent isotope ratio data in human plasma samples obtained after oral administration of a single 3.2 mg 70Zn dose. Both methods are suitable for kinetic study of plasma appearance of this isotope and each has advantages and stations. Although both methods are highly specialized requiring both significant capital outlay and personnel training, several facilities are currently available throughout the world where such work could be carried out with either analytical method.

This work was supported in part by Grant No. 79191 IZPFR from the National Science Foundation. The MIT authors gratefully appreciate the assistance of MITRII

171

and CRC staff in conducting these experiments, as well as the cooperation Center for Analytical Chemistry of the U.S. National Bureau of Standards.

of the

References I Aamodt, R.L., Rundle, W.R., Johnston, G.S., Foster, D. and Henkin, R.I. (1979) Zinc metabolism in humans after oral and intravenous administration of Zn-69m. Am. J. Clin. Nutr. 32, 559 2 Solomons, N.W., Jacob, R.A., Pineda, 0. and Viteri, F. (1979) Studies on the bioavailability of zinc in man. II. Absorption of zinc from organic and inorganic sources. Am. J. Clin. Nutr. 32, 2495 3 Janghorbani, M., Ting, B.T.G. and Young, V.R. (1980) Measurement of “Zn and “Zn in human blood in reference to the study of zinc metabolism. Am. J. Clin. Nutr. 34, 581 4 Shields, W.R. (ed.) (1970) Analytical Mass Spectrometry Section: Summary of Activities, NBS Tech. Note 546, U.S. Government Printing Office, Washington, DC 5 Gramlich, J.W., Machlan, L.A., Murphy, T.J. and Moore, L.J. (1977) The Determination of Zinc, Cadmium and Lead in Biological and Environmental Materials by Isotope Dilution Mass Spectrometry, Trace substances in environmental health-XXI, (D.D. Hemphill, ed.), University of Missouri, Columbia, MO, pp. 376-380 6 Barnes, I.L., Murphy, T.J., Gramlich, J.W. and Shields, W.R. (1973) Lead separation by anodic deposition and isotope ratio mass spectrometry of microgram and smaller samples. Anal. Chem. 45. 1881