High Precision Zinc Isotopic Measurements Applied to Mouse Organs

Summary

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Abstract

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (⁶⁴Zn, ⁶⁶Zn, ⁶⁷Zn, ⁶⁸Zn and ⁷⁰Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO₃ and H₂O₂. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO₃ medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique). This full typical procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

Introduction

The measurement of high-precision (better than 100 ppm/atomic mass unit) zinc stable isotope composition has only been possible for about 15 years thanks to the development of multi-collector plasma-source mass-spectrometers and has since been mostly applied in Earth and planetary sciences. The applications to the medical field are novel and have a strong potential as biomarkers for diseases that modify the metabolism of zinc (e.g., Alzheimer disease). This paper reports a method to measure with high precision the natural stable isotope ratios of zinc in various mouse organs. The same would be applicable to human samples. The method consists of the dissolution of the organs, the chemical purification of zinc from the rest of the atoms, and then the analysis of the isotope ratio on a mass-spectrometer.

The quality of Zn isotopic measurements is dependent on the quality of the chemical purification (purity of Zn, low blank compared to the amount of Zn present in the sample, high chemical yield of the procedure) and on the control of the instrumental bias. The high purity of the final Zn fraction is needed to remove both isobaric interferences and non-isobaric interference that create a matrix effect. Isobaric nuclides create direct interferences (e.g., ⁶⁴Ni). Non-isobaric interferences generate the so-called “matrix” effect and alter the analytical precision of the measurements by changing the condition of the ionization compared to the pure zinc standard to which the samples are compared to¹. A low blank (< 10 ng) indicates that there is no contamination of the samples by external Zn that would bias the measured isotopic composition. As Zn isotopes can be fractionated during ion-exchange chromatography², the collection of all the Zn atoms ensures that no isotopic fractionation occurs, which implies that the chemical procedure should have a full yield. Finally, the correction of the instrumental isotopic fractionation during the mass-spectrometry measurement is done via the “standard bracketing” method.

Therefore, the main difficulties to obtain precise measurements are controlling the external contamination (i.e., low blank), producing a full yield chemical purification that is clean of any other atoms or molecules, and correcting the instrumental isotopic fractionation on the mass-spectrometer. In this paper we will describe our analytical protocol to separate Zn from the mouse organs as well as the mass-spectrometry measurements.

The extraction is done using a low quantity of diluted acids (HBr/HNO₃ media) on micro-columns (0.5 µl and 0.1 µl) of anion-exchange resin. It has a full yield and the measurements have an external reproducibility better than 50 ppm on the ⁶⁶Zn/⁶⁴Zn ratio. Another advantage of the method is that it is very fast. The method is therefore very well adapted to medical sciences, in which one needs to analyze a large number of samples compared to geosciences, where these analytical methods were developed.

Protocol

NOTE: Procedures involving animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the Université Paris Diderot.

1. Preparation of Materials

1. Sub-boil distill 1 L of the acids (HNO₃, HBr) in order to purify them from impurity.
2. Clean the beakers and tip adaptor in a hot (~ 100 °C) concentrated HNO₃ acid bath for at least two days.
3. Wash the pipette tips in a cold 3 N HNO₃ bath for several days and rinse individually three times with de-ionized water.

2. Sample Preparation

1. Anesthetize the mice by intraperitoneal injection of ketamine and xylazine. Assess anesthesia by the toe pinch method.
2. Collect the blood by a cardiac puncture in the presence of heparin in 1.5 ml tubes.
3. Separate the plasma from blood cells by centrifugation (10 min, 1,500 x g) and transfer the plasma to polypropylene cryogenic vials using polypropylene tips.
4. Remove the remaining blood from organs by cutting the hepatic vein and injecting DPBS through the heart. Assess death of the mouse by cervical dislocation.
5. Harvest the organs with sterile stainless steel instruments, free them of surrounding fat if any, and snap-freeze them in polypropylene cryogenic vials.

3. Chemical Purification

1. First, dissolve the samples in a mix of ~ 1 ml of concentrated (30%) H₂O₂ and ~ 1 ml of concentrated (~ 15 M) HNO₃. Do all these steps inside a fume hood.
   1. Place the whole organ of interest into a 15 ml Teflon beaker. Then, add the H₂O₂/HNO₃ to the beaker⁵. Keep the beaker open for a few minutes in order to avoid splashes due to the reaction of oxidation of the organic matter and the release of CO₂.
   2. Finally, put the beaker on a hot plate at about 100 °C for a couple of hours or until the solution is perfectly clear.
2. Open the beaker and dry the solution on a hot plate at about 100 °C.
3. Once the sample is dry, add 1 ml of 1.5 N HBr to the samples; close the beaker and let it dissolve on a hot plate at 100 °C for a couple of hours.
4. Meanwhile prepare the 500 µl columns.
   1. Add 500 µl of the AG1X8 200-400 mesh resin to the column and put it on the column rack with a trash beaker below it. Wash the resin by alternating: 5 ml of 18.2 MΩ ⋅ cm water, 5 ml of 0.5 N HNO₃, 5 ml of water, 5 ml of 0.5 N HNO₃, and then 5 ml of water. Condition the resin with 5 ml of 1.5 N HBr.
5. Remove the beakers from the hot plate and put them in an ultrasonic bath for about 30 min, and then let the beakers cool down to RT.
6. Once the beaker is cooled and the resin is washed, open the beaker. Put the tip adaptor to the syringe, add a pipette tip; pipette the 1 ml of sample and load it onto the resin (very slowly in order not to agitate the resin).
7. Once all the liquid passes through the column, add 5 ml of 1.5 N HBr.
8. Once the 5 ml of 1.5 N HBr pass through the column, replace the trash beaker with a clean 15 ml beaker.
9. Add 5 ml of 0.5 N HNO₃ 2.5 ml at a time. At this stage the Zn is eluted from the resin.
10. Once 5 ml of HNO₃ passes through the column, remove the beaker and place it on a hot plate at 100 °C until dried.
11. Remove the column from the column holder; trash the resin (use a new resin for each sample).
12. Once the sample is dry, repeat the protocol with the same volume of acids on a smaller column (100 µl) and then place it on a hot plate until dried. The sample is now ready for mass-spectrometry.

4. Mass-spectrometry Measurement

1. Analyze the Zn isotopic composition on a multi collector-inductively coupled plasma-mass spectrometer (MC-ICP-MS).
   1. Use the machine parameters summarized in Table 1.
2. Position the Faraday cups to collect at mass (m/z) of ⁶²Ni, ⁶³Cu, ⁶⁴Zn, ⁶⁵Cu, ⁶⁶Zn, ⁶⁷Zn and ⁶⁸Zn.
3. Prepare a solution containing 500 ppb Zn in 0.1 M HNO₃ for isotopic analysis.
4. Analyse the 500 ppb solution of Zn by using a spray chamber combined with a 100 µl/min teflon nebulizer. For each sample, measure 30 scans (1 block of 30 cycles) in which the integration time of each scan is 8.389 sec.
5. Correct the background by subtracting the on-peak zero intensities from a blank solution (the 0.1 M HNO₃ solution used to re-dissolve the samples).
6. Control and correct possible ⁶⁴Ni isobaric interference by measuring the intensity of the ⁶²Ni peak. Assume that the ⁶⁴Ni/⁶²Ni ratio is natural (0.2548), correct this value from the instrumental mass bias, and then remove the ⁶⁴Ni on the mass 64 as:
   ⁶⁴Zn_real= ⁶⁴Zn_measured - ⁶⁴Ni = ⁶⁴Zn_measured - (⁶⁴Ni/⁶²Ni)_natural x ⁶²Ni_measured.
7. Correct the instrumental mass bias by bracketing each of the samples with a 500 ppb standard solution of the JMC Lyon Zn standard (or another available standard such as IRMM-3702). Perform the standard bracketing by dividing the ⁶⁶Zn/⁶⁴Zn ratio of the sample by the average of the ⁶⁶Zn/⁶⁴Zn ratio of the two standards measured before and after the sample minus 1 and multiplied by 1,000 (see Equation 1). Typical external precision on the JMC Lyon Zn standard is 0.05 permil/amu (2 standard deviation, 2 s.d.).

Results

In 1.5 N HBr, the main zinc species (ZnBr3-) forms very strong complexes with the anion-exchange resin, while most other elements do not interact with the resin. Zinc is then recovered by changing the medium to diluted HNO₃, changing the speciation of Zn to Zn²⁺ which is released from the resin^6,7.

Isotope ratios are typically expressed as parts per 1,000 deviations relative to a standard:

Discussion

The reproducibility of the measurements is evaluated through replicated analyses of the same samples carried out during different analytical sessions. For example⁶, we have replicated the same terrestrial rock 7 times and we obtained the results reported in the Table 2.

As expected from the theory of isotopic fractionation¹⁰ and as measured in any solar system material so far (e.g., meteorite^11-13, plants^3-5, deep-sea s...

Materials

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Multi-collection inductively-coupled-plasma mass-spectromter	Thermo-Fisher
Anion-exchange resin AG1 X8 200-400	Bio-Rad	140-1443-MSDS
teflon beakers	Savillex	200-015-12
Home-made teflon colunms made with shrinkable teflon