Speciation of protein-binding zinc and copper in human blood serum by chelating resin pre-treatment and inductively coupled plasma mass spectrometry

Abstract

A method for the speciation of zinc and copper binding with proteins in human serum was explored by chelating resin (Chelex-100) pre-treatment and inductively coupled plasma mass spectrometry (ICP-MS). It was shown by a SEC (size-exclusion chromatography)-ICP-MS system that albumin-zinc and albumin-copper (loosely-bound species) could be selectively removed from serum by adsorption on the Chelex-100 resin after the chelating resin pre-treatment, while α₂-macroglobulin-zinc and ceruloplasmin-copper (firmly-bound species) remained in the serum. The zinc and copper bound with α₂-macroglobulin and ceruloplasmin, respectively, were then determined by ICP-MS after batch treatment of the serum samples with the Chelex-100 resin. In addition, the total concentrations of zinc and copper were also determined by ICP-MS after a 20-fold dilution with 0.1 M HNO₃. The albumin-zinc and -copper were estimated as the differences between the concentrations of total and firmly-bound species. The present batch pre-treatment method was applied to the speciation analysis of zinc and copper binding with proteins in sera donated from 25 healthy volunteers as well as from a pregnant woman and a myelodysplastic syndrome patient. The observed concentrations of α₂-macroglobulin-zinc and ceruloplasmin-copper were in the ranges 109–202 ng ml⁻¹ (12.4–31.3% of total zinc) and 513–880 ng ml⁻¹ (90.6–99.7% of total copper), respectively. The present method is simple (only addition of the chelating resin and centrifugation is required) and reproducible (average RSD = 2% for α₂-macroglobulin-zinc and 1% for ceruloplasmin-copper in intra-assay measurements, and 5% for α₂-macroglobulin-zinc and 4% for ceruloplasmin-copper in inter-assay measurements), and there is less risk of contamination during separation.

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Introduction

Zinc and copper are known to be essential trace elements in man and animals.^1,2 Thus, the determination of zinc and copper in human blood and organs is important in medical diagnosis.³ In clinical analysis, the total concentrations of zinc and copper in serum, which are often referred to as TSZn (total serum-zinc) and TSCu (total serum-copper), respectively, are generally used as convenient parameters for medical diagnoses of health and disease.^4,5 However, the biological activities or toxicities of trace elements strongly depend on their chemical forms in blood serum, and thus data for their total concentrations are not sufficient for medical diagnosis. Therefore, speciation analyses of zinc and copper species in human blood serum are now receiving great interest not only in medical diagnosis, but also in biological and environmental sciences.⁶

Most of the zinc and copper in blood serum is bound with proteins,^7–10 although about 1% exists as amino acid complexes.^11–13 It is also known that more than 90% of copper is firmly bound with ceruloplasmin,^7,8 while the remainder, the labile pool, is loosely bound with albumin and amino acids. On the other hand, zinc is loosely bound mainly with albumin, and a small amount is firmly bound with α₂-macroglobulin.^9,10 Various techniques such as sucrose density gradient centrifugation,¹⁴ polyethylene glycol precipitation,¹⁵ electrophoresis,¹⁶ chromatography,^17–22 and ultrafiltration^23–25 have been employed for the separation of metal binding species in human blood serum. More recently, the chromatographic separation of trace elements including zinc and copper has been investigated by using an HPLC-ICP-MS system.^26–28 Since zinc or copper bound with albumin is labile, their adsorption on materials used in such separation techniques often causes serious problems in accuracy and precision.^{14,17,18,21,22} Therefore, cleaning of the experimental equipment and prevention of adsorption of analytes on the equipment during the separation process have inevitably been required. The aim of the present paper was to explore a convenient separation method for the speciation of protein-bound zinc and copper, utilizing their different binding abilities with proteins in serum, where ICP-MS was used as a highly sensitive determination method.

In previous papers,^23–25 EDTA was employed for the assay of exchangeable zinc and copper in human blood serum. In these studies, zinc or copper loosely-bound with albumin and amino acids in serum produced EDTA complexes through an exchange reaction, and the EDTA complexes were separated from α₂-macroglobulin-zinc and ceruloplasmin-copper (firmly-bound metal species) by an ultrafiltration method. However, contamination from and adsorption on the ultrafiltration filter is often very serious, and tedious washing of the filter must inevitably be performed. In the present experiments, the Chelex-100 resin, which has similar functional groups to EDTA, was employed as an adsorbent of loosely-bound zinc and copper. Selective removal of loosely-bound zinc and copper in serum after pre-treatment with the Chelex-100 resin was experimentally assured by a SEC (size-exclusion chromatography)-ICP-MS system. In practice, zinc and copper loosely bound with albumin and amino acids in blood serum were selectively collected by adsorption on the Chelex-100 resin in a batch method and removed by centrifugation. Then, the firmly-bound zinc and copper remaining in the serum were determined by ICP-MS.

Experimental

Instrumentation

A Model SPQ 8000A ICP-MS instrument (Seiko Instruments, Chiba, Japan), composed of a quadrupole mass spectrometer, was used for the determination of zinc and copper at m/z 66 and 65, respectively. Since polyatomic ions of ³²S³²S and ⁴⁰Ar²³Na often cause spectral interferences with ⁶⁴Zn and ⁶³Cu, respectively,²⁹⁶⁶Zn and ⁶⁵Cu are preferable in the analysis of blood serum, which contains a large amount of sulfur and sodium.

The instrumental operating conditions for ICP-MS are summarized in Table 1. For the chromatographic measurements, a higher rf incident power was applied to the plasma, compared with ICP-MS with conventional pneumatic nebulization, because of the load of organic buffer and proteins in the mobile phase and serum samples, respectively. The reflected power was less than 5 W in both cases, and no significant stability change was observed. Matrix effects and instrumental drift in the ICP-MS measurements were corrected by the internal standard method using Rh as the internal standard element. Usually, Ge (m/z 74) is used as the internal standard element in the determination of zinc and copper by ICP-MS. However, the polyatomic ion of ³⁷Cl³⁷Cl is prone to interfere with ⁷⁴Ge,³⁰ when a large amount of chlorine is present in the sample, such as in blood serum. Thus, Rh was chosen as the internal standard element, which was not detected in blood serum. The internal standard correction was carried out in a similar manner to that described in previous papers. ^31,32

Table 1 Operating conditions of ICP-MS and SEC-ICP-MS hyphenated system

a The conditions not described here are the same as those in conventional ICP-MS.
ICP-MS—	Seiko SPQ 8000A
Plasma conditions:
Rf frequency	27.12 MHz
Reflected power	< 5 W
Incident rf power	1.0 kW
Outer gas flow rate	Ar 16 l min^−1
Intermediate gas flow rate	Ar 1.0 l min^−1
Carrier gas flow rate	Ar 1.0 l min^−1
Sampling conditions:
Sampling depth	12 mm from load coil
Sample uptake rate	0.8 ml min^−1
Data acquisition:
Scanning mode	Peak hopping
Data points	3 points per peak
Dwell time	10 ms per point
Integration	100 times
SEC / ICP-MS hyphenated system—
SEC—
Column	Superose 12HR
Mobile phase	0.1 M Tris-HCl buffer + 2.5 mM CaCl2 (pH 7.4)
Flow rate	0.4 ml min^−1
Injection volume	200 μl
ICP-MS^a—
Plasma conditions:
Incident rf power	1.3 kW
Reflected power	< 5 W
Carrier gas flow rate	Ar 0.8 l min^−1
Sampling conditions:
Sampling depth	10 mm from load coil
Sample uptake rate	0.4 ml min^−1
Data acquisition:
Data points	1 point per peak
Dwell time	100 ms per point
Total measurement time	5400 s

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Chemicals

The standard solutions of zinc and copper were prepared from commercially available single-element standard solutions (1000 μg ml⁻¹) for atomic absorption spectrometry (Wako Pure Chemicals, Osaka, Japan). Ammonia, acetic acid, and hydrochloric acid used were of electronics industry grade, purchased from Kanto Chemicals (Tokyo, Japan). Pure water was used throughout the experiments and was prepared by a Milli-Q water purification system (resistivity 18 MΩ cm, Nihon Millipore Kogyo, Tokyo, Japan).

The Chelex-100 resin (sodium form, 200–400 mesh) used as the adsorbent of loosely-bound zinc and copper was obtained from Bio-Rad Laboratories (Richmond, CA, USA). The resin was cleaned before use by keeping it in 5 M HCl overnight. After washing with pure water, the resin was converted to the NH₄⁺ form by preconditioning with 1 M ammonium acetate solution (pH 7.4).

Blood serum samples

The human blood samples were collected from 25 healthy volunteers with a stainless-steel needle (coated with silicone) fitted to a 30 ml poly(propylene) syringe, and were transferred into silicone-coated glass tubes. After clotting, the blood samples were centrifuged at 3000 rpm for 20 min. The supernatants were used as the serum samples. To minimize metal contamination, poly(propylene) syringes and silicone-coated glass tubes were soaked in 2 M HCl for 2 d, rinsed with pure water and dried before use.

In the determination of total zinc and copper in blood serum by ICP-MS, the serum samples were diluted 20-fold with 0.1 M HNO₃.

Batch treatment of blood serum with the chelating resin for separation of loosely- and firmly-bound species

In the determination of firmly-bound zinc (α₂-macroglobulin) and copper (ceruloplasmin) by ICP-MS, the blood serum samples were pre-treated with the chelating resin in a batch method, as follows. First, 1 ml of blood serum sample was taken in a centrifuge tube (Corning Glass, Corning, NY, USA), into which 0.4 g of Chelex-100 resin was added. After shaking gently for ca. 1 min, the serum sample was set aside for 5 h at room temperature, and then centrifuged at 3000 rpm for 1 min. A 0.1 ml aliquot of the supernatant was transferred into a poly(propylene) tube and diluted with 2 ml of 0.1 M HNO₃, which contained an internal standard element (Rh, 10 ng ml⁻¹) for matrix effect correction in the ICP-MS measurement. The resin-treated serum samples were analysed for α₂-macroglobulin-zinc and ceruloplasmin-copper by ICP-MS without further pre-treatment.

In this separation process, the centrifuge and poly(propylene) tubes were cleaned before use in a similar manner to the syringes and glass tubes used previously.

Chromatographic measurements of protein-binding zinc and copper in serum

In order to demonstrate selective removal of loosely-bound zinc and copper from human blood serum by the batch pre-treatment with the chelating resin, the element-selective chromatograms for protein-binding zinc and copper in serum before and after the chelating resin pre-treatment were recorded by a SEC-ICP-MS hyphenated system. The present SEC-ICP-MS system was similar to those used for speciation analysis of trace elements in natural water, reported previously.^33,34 The operating conditions of the SEC-ICP-MS system are also summarized in Table 1. The SEC-ICP-MS system consisted of a high-pressure pump (Model LC-10AD, Shimadzu, Kyoto, Japan), a sample injector (Model V7, Pharmacia-LKB, Uppsala, Sweden) with a 200 μl sample loop, and a SEC column (Superose 12HR, Pharmacia-LKB). The SEC column used had a molecular weight permeation range of 3000–200000 Da. The molecular weight calibration of the SEC column was performed by using the following standard materials: blue dextran (2000000), β-amylase (200000), albumin (66000), carbonic anhydrase (29000), cytochrome (12400), and aprotinin (6500), where the numbers in parentheses indicate the molecular weight of each compound in Da. A 0.1 M Tris-HCl buffer solution (pH 7.4) was used as a mobile phase, to which 2.5 mM calcium chloride was added to prevent adsorption of zinc and/or copper on the SEC column. The flow rate of the mobile phase was 0.4 ml min⁻¹, which was the optimized separation condition for the present SEC column. Since the ICP-MS instrument was used as an element-selective detector, the eluate from the SEC column was introduced into the nebulizer of the ICP-MS via Teflon tubing. Polyatomic ion interference due to the constituents of the mobile phase was not observed.

Results and discussion

Determination of total concentrations of zinc and copper in blood serum reference material

In the present work, the total concentrations of zinc and copper in the blood serum samples were determined by ICP-MS after a 20-fold dilution with 0.1 M HNO₃. In order to ensure the validation of the analytical values obtained, zinc and copper in Second Generation Blood Serum Reference Material issued by Ghent University (Belgium) were determined by ICP-MS. The results are shown in Table 2, together with the data obtained by acid digestion³⁵ and the certified values.³⁶ As is seen in Table 2, the total concentrations of zinc and copper in the blood serum reference material obtained by the present dilution method are in good agreement with the literature values³⁵ and certified values³⁶ within the standard deviations in each experiment. The present 20-fold dilution method is simple and results in less contamination during sample treatment and preparation because only 0.1 M HNO₃ is added to the serum samples. Thus, in the following experiments, the total concentrations of zinc and copper in all blood serum samples collected from volunteers were determined by ICP-MS after a 20-fold dilution.

Table 2 Analytical results for the determination of total concentrations of zinc and copper in Second Generation Blood Serum Reference Material (Ghent University) after dilution

Element	Observed value^a/ng ml^−1	Literature value^b/ng ml^−1	Certified value^c/ng ml^−1
a Mean ± s (standard deviation), n = 3, where the serum sample was diluted 20-fold with 0.1 M HNO3.b Sample was digested with concentrated HNO3, and diluted 10-fold with 0.1 M HNO3. Cited from ref. 35.c Cited from ref. 36.
Zn	880 ± 17	880 ± 18	873 ± 35
Cu	1020 ± 12	975 ± 8	1010 ± 40

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Selective separation of loosely- and firmly-bound zinc and copper in blood serum by chelating resin pre-treatment

Selective removal of loosely- and firmly-bound zinc and copper in blood serum by the chelating resin batch treatment was examined by the SEC-ICP-MS system. The SEC traces for zinc and copper in human blood serum before and after the chelating resin pre-treatment are shown in Fig. 1 and 2, respectively, which were obtained by ICP-MS detection at m/z 66 (zinc) and m/z 65 (copper). The calibration graph for molecular weights is also shown in each figure. In this experiment, 0.4 g of Chelex-100 resin was added to 1 ml of serum, and the mixture was set aside for 24 h. After centrifugation, the supernatant was subjected to chromatographic measurement with detection by ICP-MS. Since the physiological pH of blood serum is usually 7.4 ± 0.2, the pH of the mobile phase was adjusted to 7.4 by using Tris buffer solution.

Fig. 1 Element-selective chromatograms for zinc in human blood serum samples obtained by the LC-ICP-MS system before (light line) and after (bold line) chelating resin pre-treatment. Peak A, α₂-macroglobulin-zinc (MW: 700000 Da). Peak B, albumin-zinc (MW: 68000 Da). The solid line with circle symbols is the molecular weight calibration graph for the SEC column.

Fig. 2 Element-selective chromatograms for copper in human blood serum samples obtained by the LC-ICP-MS system before (light line) and after (bold line) chelating resin pre-treatment. Peak C, ceruloplasmin-copper (MW: 150000 Da). Peak D, albumin-copper (MW: 68000 Da). The solid line with circle symbols is the molecular weight calibration graph for the SEC column.

In Fig. 1, two peaks can be seen at elution volumes of about 8 ml (Peak A) and 12 ml (Peak B) before the chelating resin pre-treatment, although the elution volume of 8 ml corresponds to the void volume of the SEC column. From the calibration of the SEC column using α₂-macroglobulin (MW: 700000 Da) and albumin (MW: 68000 Da), these two peaks were attributed to zinc bound with α₂-macroglobulin and albumin, respectively, which are hereafter referred to as α₂-macroglobulin-zinc and albumin-zinc. It should be noted that Peak B disappeared from the chromatogram (light line in Fig. 1) after the chelating resin pre-treatment. These results indicate that albumin-zinc was selectively removed from blood serum by the chelating resin pre-treatment because of the loose binding of zinc with albumin.

In Fig. 2, one peak with a small shoulder peak is apparently observed for copper at an elution volume of about 10.5 ml, where the retention volume of the small shoulder peak is ca. 12 ml. According to a preliminary study using ceruloplasmin and albumin, it was found that the main peak (Peak C) could be assigned to copper bound with ceruloplasmin (MW: 150000 Da), and the small shoulder peak (Peak D) to copper bound with albumin (MW: 68000 Da). These species are also hereafter referred to as ceruloplasmin-copper and albumin-copper. Since the amount of albumin-copper in serum was much less than the amount of ceruloplasmin-copper, the peaks for these two proteins could not be separated by the present SEC column. However, it is seen in Fig. 2 that the shoulder peak for albumin-copper eluting at ca. 12 ml almost disappeared after the chelating resin pre-treatment. These results suggest that ceruloplasmin-copper is the main species in serum, and albumin-copper can be removed by the chelating resin. Consequently, zinc and copper loosely bound with albumin can be selectively separated from α₂-macroglobulin-zinc and ceruloplasmin-copper (firmly-bound species) in serum by the present chelating resin pre-treatment method, followed by separation of the resin from the blood serum by centrifugation. Thus, speciation of zinc and copper in blood serum by the chelating resin pre-treatment and ICP-MS was carried out by the batch method in the following experiments.

Optimum conditions for chelating resin pre-treatment

The amount of chelating resin necessary to separate albumin-zinc and -copper from α₂-macroglobulin-zinc and ceruloplasmin-copper in blood serum was investigated to optimize the separation conditions. In the following optimization experiment, including the resin treatment time, the pH was kept at 7.4, viz., that of serum itself (no optimization), to avoid denaturation of proteins. Fig. 3 shows the concentrations of zinc and copper remaining in the blood serum after pre-treatment with different amounts of the chelating resin, where 1 ml of the serum sample was used and the reaction time in the chelating resin pre-treatment (referred to as ‘resin treatment time’) was 24 h at room temperature. As is seen in Fig. 3, the concentration of zinc remaining in the serum decreased with an increase in the resin amount, and became almost constant above 0.3 g of the resin. Similarly, the concentration of copper remaining in the blood serum was also almost constant above 0.3 g of the resin. These results indicate that 0.4 g of the resin was sufficient to adsorb almost completely albumin-zinc and -copper in serum on the chelating resin.

Fig. 3 Effect of resin amount on residual concentrations of zinc and copper in human blood serum. ●: Copper, ○: zinc. Conditions for the chelating resin pre-treatment: Chelex-100 resin was added to 1 ml of serum and set aside for 24 h.

Optimization of the resin treatment time was also performed by measuring the concentrations of zinc and copper remaining in the blood serum, where 1 ml of serum was treated with 0.4 g of the resin in the batch method. The result is shown in Fig. 4. When the resin treatment time was longer than 5 h, the concentrations of zinc and copper remaining in the blood serum became constant, and were almost at the same concentration levels as those obtained in Fig. 3. These results indicate that the exchange reactions of zinc and copper between albumin and the chelating resin effectively reached equilibrium after 5 h. Hence, 0.4 g of the Chelex-100 resin and a resin treatment time of 5 h were chosen as the optimum conditions.

Fig. 4 Effect of resin treatment time on residual concentrations of zinc and copper in human blood serum. ●: Copper, ○: zinc. Conditions for the chelating resin pre-treatment: 0.4 g of Chelex-100 resin was added to 1 ml of serum.

Determination of α₂-macroglobulin-zinc and ceruloplasmin-copper in blood serum by the chelating resin pre-treatment method

The determination of α₂-macroglobulin-zinc and ceruloplasmin-copper in human blood serum by ICP-MS after the chelating resin pre-treatment was carried out. The serum sample used was one of the 25 samples collected from healthy volunteers, details of which will be given later. The total concentrations of zinc and copper in the serum sample were also determined by ICP-MS after diluting 20-fold with 0.1 M HNO₃, as mentioned earlier. The analytical results are summarized in Table 3, together with the relative standard deviations (RSDs) of the observed values estimated from three replicate analyses. In Table 3, the analytical results for intra-assay (three replicate measurements on a single day) and inter-assay (three replicate measurements on each of three days) measurements are shown for comparison. As is seen in Table 3, α₂-macroglobulin-zinc and ceruloplasmin-copper accounted for 20.7% of the total zinc and 92.4% of the total copper in serum, respectively, for the intra-assay measurements. These values for firmly-bound zinc and copper are reasonable, compared with those reported previously^13–23 as well as those obtained in the following experiment (Table 4). The RSD of each observed value in the intra-assay measurements was below 2%, while that in the inter-assay measurements was below 5%. Thus, the present chelating resin pre-treatment method is considered to be sufficiently precise for the quantification of α₂-macroglobulin-zinc and ceruloplasmin-copper in human blood serum.

Table 3 Analytical results for the concentrations of total and firmly-bound zinc and copper in human blood serum

Intra-assay^a		Inter-assay^b
Observed values^c/ng ml^−1	RSD^d (%)	Observed values^c/ng ml^−1	RSD^d (%)
a Three replicate analyses on a single day.b Three replicate analyses on three different days.c Mean ± s (n = 3); the values in parentheses indicate the ratios of α2-macroglobulin-zinc and ceruloplasmin-copper to total zinc and copper, respectively.d Relative standard deviation was evaluated from three replicate analyses.
Total zinc	615 ± 12	2	621 ± 26	4
α2-Macroglobulin-zinc	127 ± 3	2	130 ± 6	5
(20.7%)	(20.9%)
Total copper	766 ± 9	1	755 ± 35
Ceruloplasmin-copper	708 ± 8	1	703 ± 32	4
(92.4%)	(93.1%)

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Table 4 Analytical results for total, loosely-, and firmly-bound zinc and copper in human blood serum collected from 25 healthy volunteers, a pregnant woman and a myelodysplastic syndrome patient

Observed value		Pregnant woman^c	Patient^d
Concentration^a/ng ml^−1	Ratio^b (%)	Concentration/ng ml^−1	Concentration/ng ml^−1
a Mean ± s (n = 25: male 14, female 11). The values in parentheses indicate the concentration range for sera collected from 25 healthy volunteers.b Ratio (%) of loosely- or firmly-bound metal to the total concentration.c 20 weeks into pregnancy.d Myelodysplastic syndrome patient.e The concentration was calculated by subtracting the concentration of α2-macroglobulin-zinc or ceruloplasmin-copper from that of total zinc or copper.
Total Zn	701 ± 123 (465–988)	397	300
α2-Macroglobulin-zinc	138 ± 28  (109–202)	20.2 ± 4.3 (12.4–31.3)	132	74
Albumin-zinc^e	562 ± 118 (342–866)	79.8 ± 4.3 (68.7–87.6)	265	226
Total Cu	756 ± 81  (566–902)	1440	870
Ceruloplasmin-copper	723 ± 77  (513–880)	95.7 ± 2.8 (90.6–99.7)	1410	813
Albumin-copper^e	32 ± 22  (2–73)	4.3 ± 2.8 (0.3–9.4)	30	57

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The blank values of zinc and copper in the present method were estimated by using 1 ml of pure water, where the same chelating resin pre-treatment as that for blood serum was carried out. All the experimental procedures in the estimation of the blank values, as well as in the chelating resin pre-treatment, were performed in a clean bench (Class 100). In addition, all the experimental equipment was cleaned by soaking in 2 M HCl for a few days, as mentioned earlier. As a result, the blank values of zinc and copper were below the instrumental detection limits (zinc 0.5 ng ml⁻¹, copper 1 ng ml⁻¹) obtained by the present ICP-MS instrument, which were estimated as their concentrations corresponding to three times the standard deviation (3ς) of the blank signal intensities (n = 10), when 0.1 M HNO₃ solution was nebulized as the blank. These results indicate that the present method is almost free from contamination by zinc and copper in the separation process. Consequently the present chelating resin pre-treatment method followed by ICP-MS measurement is useful for the specific quantification of α₂-macroglobulin-zinc and ceruloplasmin-copper in human blood serum.

Distributions of loosely- and firmly-bound zinc and copper in blood serum

The present chelating resin pre-treatment method was applied to the determination of α₂-macroglobulin-zinc and ceruloplasmin-copper in blood serum samples collected from 25 healthy volunteers, a pregnant woman (20 weeks into pregnancy), and a myelodysplastic syndrome patient. The analytical results are summarized in Table 4. In Table 4, the total concentrations of zinc and copper were also obtained by ICP-MS, as described earlier, and the concentrations of albumin-zinc and -copper (loosely-bound species) were calculated by subtracting the concentrations of α₂-macroglobulin-zinc and ceruloplasmin-copper from their total concentrations. Since zinc and copper bound with amino acids, which may also be loosely-bound species, are about 1% of their total concentrations in serum,^{12–14,23–25} the concentrations of amino acid-bound zinc and copper were neglected in the estimation of loosely-bound zinc and copper in Table 4. It is seen in Table 4 that, for healthy volunteers, the ratios of α₂-macroglobulin-zinc and albumin-zinc to the total zinc concentration are 20.2 ± 4.3% (12.4–31.3%) and 79.2 ± 4.3% (68.6–87.7%), respectively, and that those of ceruloplasmin-copper and albumin-copper to the total copper concentration are 95.7 ± 2.8% (90.6–99.7%) and 4.4 ± 2.8% (0.3–9.4%), respectively.

The analytical results for blood serum samples from a pregnant woman and a myelodysplastic syndrome patient are also shown in Table 4. The total concentration of zinc in the serum of the pregnant woman was significantly lower than the mean value for healthy volunteers, although the concentrations of α₂-macroglobulin-zinc were not very different from each other. Thus, it is seen that the decrease of total zinc is caused by the decrease of albumin-zinc in pregnancy. On the other hand, the total concentration of copper in the pregnant woman was almost 2-fold higher than the average value for healthy volunteers. It is seen from Table 4 that such an increase of the total copper concentration in the pregnant woman was caused by the increase of ceruloplasmin-copper. For the myelodysplastic syndrome patient, the total concentration of zinc was also distinctly low, where the concentrations of both albumin-zinc and α₂-macroglobulin-zinc were much lower than those of healthy volunteers. In contrast, the total concentrations of copper and its protein-binding species were not very different from those for healthy volunteers. The status of copper in the myelodysplastic syndrome patient was apparently different from that in the pregnant woman.

A comparison of the concentration distributions of total, loosely- and firmly-bound zinc in serum obtained by the present method with those obtained by other methods is summarized in Table 5. In Table 5, the ratios (%) of albumin- and α₂-macroglobulin-copper to total zinc are also shown in parentheses. It is well known that the concentrations of trace elements in serum are influenced by lifestyle, geographic, and demographic conditions, and thus the concentrations of trace elements are different in each population.^5,37 In fact, the observed mean concentrations of total-zinc and albumin-zinc in the Japanese group examined here were lower than the reported values.^15,20,22,24 However, the observed mean concentration of α₂-macroglobulin-zinc was similar to the reported values. These results suggest that the variations in total serum zinc concentrations may be attributed mainly to the concentration changes or differences of albumin-zinc in the present group.

Table 5 Comparison of analytical results for total, albumin-bound, and α₂-macroglobulin-bound zinc in human blood serum obtained by various methods

Concentration/ng ml^−1^a
This work (n = 25)	Polyethylene glycol precipitation^b (n = 20)	Affinity chromatography^c (n = 123)	Gel filtration–ion-exchange chromatography^d (n = 10)	Ultrafiltration^e (n = 20)
a Mean ± s; the values in parentheses are the ratios (%) of loosely- or firmly-bound species to the total concentration.b Cited from ref. 15.c Cited from ref. 20.d Cited from ref. 22.e Cited from ref. 24.f The concentration was calculated by subtracting the concentration of α2-macroglobulin-zinc from that of total zinc.
Albumin-zinc	562 ± 118^f	679 ± 83	794 ± 141	787 ± 38	775 ± 124
(80.2%)	(79.8%)	(83.8%)	(87.3%)	(78.1%)
α2-macroglobulin-zinc	137 ± 28	156 ± 27	154 ± 38	94 ± 3	197 ± 14
(19.5%)	(18.3%)	(16.3%)	(10.4%)	(19.9%)
Total zinc	701 ± 123	851 ± 91	947 ± 154	902 ± 45	992 ± 190

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The comparison data for the concentration distributions of total, loosely- and firmly-bound copper in serum are summarized in Table 6. The mean concentration of total copper in blood serum for the present Japanese group was also lower than those reported previously.^22,23 Nevertheless, the relative distribution of ceruloplasmin-copper (95.6% on average) in total serum copper obtained in the present work was similar to those obtained by other speciation methods. These results indicate that the variations of total serum copper concentrations are caused by a lowering of the concentration level of ceruloplasmin-copper, i.e., firmly-bound copper.

Table 6 Comparison of analytical results for total, albumin-bound, and ceruloplasmin-bound copper in human blood serum obtained by various methods

Concentration/ng ml^−1^a
Present method (n = 25)	Gel filtration–ion-exchange chromatography^b (n = 10)	Ultrafiltration^c (n = 20 )
a Mean ± s, the values in parentheses are the ratios (%) of loosely- or firmly-bound species to the total concentration.b Cited from ref. 22.c Cited from ref. 23.d The concentration was calculated by subtracting ceruloplasmin-copper from the total concentration of copper.
Albumin-copper	33 ± 22^d (4.4%)	98 ± 23 (8.2%)	74 (5.4%)
Ceruloplasmin-copper	723 ± 77 (95.6%)	1050 ± 110 (88.2%)	1273 (93.6%)
Total copper	756 ± 81	1190 ± 137	1360 ± 210

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Since different serum samples collected in different areas and populations were used for the results summarized in Tables 5 and 6, it is difficult to discuss any medical or biological significance from the data. However, the concentrations and distributions of total and protein-binding zinc and copper may provide important information for medical diagnosis. Therefore, it would be desirable to promote further research on the speciation of zinc and copper using common blood serum samples under an international cooperation program.

Conclusion

A chelating resin batch pre-treatment method has been established for the speciation of firmly- and loosely-bound zinc and copper in human blood serum. In the present experiments, ICP-MS was used for the determination of zinc and copper. Of course, other analytical methods such as ICP-AES and ETAAS are available for the determination of zinc and copper in combination with the chelating resin pre-treatment method.

According to the results obtained here for a pregnant woman and a myelodysplastic syndrome patient, the total concentration of zinc decreased in both patients, while total copper distinctly increased in the pregnant woman and slightly increased in the myelodysplastic syndrome patient. Under these situations, albumin-zinc distinctly decreased and ceruloplasmin-copper drastically increased during pregnancy. For the myelodysplastic syndrome patient, both albumin- and α₂-macroglobulin-zinc distinctly decreased, while albumin- and ceruloplasmin-copper showed little if any change. Since the data for pregnant and myelodysplastic syndrome patients are very limited, it is difficult to make any general or conclusive remarks from the experimental results in Table 4. However, the results may be useful for medical diagnosis.

The present speciation analysis method involving chelating resin pre-treatment and ICP-MS may be applicable to the quantification of loosely- and firmly-bound zinc and copper in human blood serum for clinical analysis and medical diagnosis.

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