Trace Metals

The toxicity of metals, both heavy metals and essential minerals, is a topic of great interest to many biomedical scientists and an issue of great public health importance. An ever increasing literature has pointed to adverse effects of early life exposure to heavy metals such as lead (Pb) and arsenic (As) on chronic disease health outcomes later in life. At the same time we continue to learn more about the health hazards of excess exposure to required elements such as manganese (Mn). The primary purpose of the Trace Metals Facility Core is to provide investigators the ability to obtain analyses of biological samples for a broad array of metals including: lead, mercury, arsenic, iron, manganese, cadmium, copper, zinc, chromium, sodium, cobalt, platinum, potassium and others.

Numerous investigators have a need for the analysis of trace metals to conduct their research in environmental health sciences. The objective of the Trace Metals Core Lab is to enable these investigators to obtain the required measurements of metals in biological samples in a scientifically valid and cost-effective manner. This Core Laboratory provides method development for these metal analyses, standardization, and quality control.  Biochemical analyses that help in the assessment of the physiological status of the subjects that are exposed to these metals are also conducted. 

The Trace Metals Facility Core enables CEHNM members to precisely measure a broad range of metals and metalloids in biological samples, including Pb, Hg, As, Se, Cd, Mn, Fe, Cu, Zn, Cr, Co, Pt, K and others. The Facility Core also provides for the measurement of major proteins of metal metabolism in serum or plasma. Concentrations of such proteins have evolved as increasingly important biomarkers of neurodegenerative, immunologic and cancer-related disease processes. The protein assays currently in place include lactoferrin, transferrin, transferrin receptor, ferritin, ceruloplasmin and others.  The lab has also provided for the measurement of serum delta-aminolevulinic acid, a surrogate marker of Pb exposure that has proven to be very useful in large historical birth cohort studies of schizophrenia for which whole blood is unavailable.  This Core provides instrumentation, resources, and expertise to scientists conducting research concerning the role of metals and metalloproteins in disease processes.

Core Director: jg24 [at] (Joseph Graziano), PhD

Core Laboratory Manager:  vns1 [at] (Vesna Slavkovich), MS

Core Service Costs

Description of Services

Investigators wishing to use the Trace Metals Core first contact Dr. Graziano to discuss their research projects and analytical needs. Information is obtained regarding the type and quantity of the biological samples to be collected, and the analyses that will be conducted. Recommendations are then made as to the type of collection vessel to be used, collection method and subsequent sample storage and shipment to the Facility Core. For some studies, a preservative is added to the collection vessel prior to sample collection and for some, the containers need to be acid-washed before being shipped to the field.

Appropriate arrangements are made when samples need to be processed in the field before they are shipped to the Core. When serum or plasma samples are required, for example, the investigator is told that separation of the serum or plasma must be done as soon as possible, before shipment of the sample, to avoid hemolysis of the blood. In this case the Core advises the investigator as to the appropriate blood collection tube and the proper technique for blood separation. For large studies, the Trace Metal Facility Core provides appropriate numbers of preprinted bar code labels to the investigator using either random digit numbers or specific coding selected by the investigator. These labels are used for sample collection tubes, tracking forms and records collected by the investigator such as questionnaires. Often, in smaller studies, the investigator’s study codes are used.

Trace Metal Analyses  

Sample Collection and Storage

Containers for the collection of urine samples are acid washed and bar-coded in the Trace Metals Lab prior to shipment to each investigator’s study site. Blood sample tubes for trace metal analysis are also provided by this lab, which routinely obtains large uniform batches which are checked for contamination prior to use.

Preanalytical Quality Control

There are many ways in which biological samples can be contaminated through the use of unsuitable collection instruments or tissue containers, blood collection vials and contaminated anticoagulants. The potential pitfalls associated with trace metal analysis are well known to laboratories like our own, with decades of experience. To prevent the possibility of contamination of blood, urine or tissue specimens for a research study, all specimen collection supplies are pre-tested for the metal of interest by the Trace Metals Core Facility, or acid-washed. We typically purchase large lots of blood collection tubes, often enough to conduct an entire research project. The purchase is made only after testing a sub-sample of the lot, provided in advance by the vendor. This has been our standard operating procedure for several large international studies, for which we pre-test and ship tissue specimen containers in advance.

Standardization and Analytical Quality Control

We participate in quality control programs for blood Pb (BPb), urinary As and Pb, and quality control programs for an array of elements run by ICP-MS-DRC. We joined the Blood Lead Laboratory Reference System, run by the Centers for Disease Control and Prevention (CDC), in 1985. We have an outstanding record and have been certified by the Occupational Safety & Health Administration (OSHA). For example, in the past three years, the agreement statistic (intraclass correlation) between the expected and observed BPb was 0.99. A unique quality control program for urinary As, run by the Institut de Sante Publique du Quebec, provides three "unknowns" every two months to laboratories throughout North America and Europe. 

We joined this program in January of 1999, but have many more years of experience with it with regard to urinary Hg analyses. The interclass correlation coefficient for total urinary As in the past five years was 0.97, showing excellent agreement with target values. Another control program that we joined in the fall of 2004, run by the same Quebec Institute, is the External Quality Assessment Scheme (QMEQAS). Three times per year they provide a set of blood, urine, serum and either hair or nail samples, with 23 different elements of “unknown” concentration, for ICP-MS work validation. Since we joined, we have been able to analyze most of the sets (unless the instrument is in speciation mode) of samples for elements of interest for our work, and the correlation is 0.99.         

Analytical Methods

We currently utilize well-established, published GFAA and ICP-MS-DRC methods, and sometimes refine them in our laboratory. With regard to past and future work, a few standard methods are in use. BPb is analyzed by the Graphite Furnace method of Fernandez and Hilligoss,1 while total urinary As is measured by the method of Nixon et al.2  Blood and serum As, Se, Mn and Pb are analyzed by ICP-MS-DRC with a method developed by modifying a few established techniques. The preparation of blood samples according to Stroh3 was modified based on suggestions from Laboratory for ICP-MS Comparison Program Institut de Sante  Publique du Quebec, while instrument parameters were established using the methods of Pruszkowski et al4 and  Nixon et al.5  When developing or adapting new GFAA and ICP-MS-DRC methods, we rely on the method of additions, and the use of appropriate Quebec or NIST standards to validate and standardize the method.

Another utilization of ICP-MS-DRC has been for the speciation of As metabolites in urine samples.  ICP-MS-DRC was coupled to HPLC and used as a detector for six major As metabolites, chromatographically separated on an anion exchange column (Hamilton PRP-X100) with 10 mM Ammonium nitrate/Ammonium phosphate, pH 9.4, as the mobile phase.6  Excellent separation by HPLC, coupled with very low detection limits of ICP-MS-DRC, allow us to detect arsenocholine (AsC), arsenobetaine (AsB), monomethyl arsenic (MMA), dimethylarsenic (DMA), arsenite (AsIII) and arsenate (AsV) (without on-line digestion of organic forms) with great precision, even in urine samples with total As concentrations as low as 10 μg/L (Figure IX-2). ICP-MS-DRC is particularly convenient for As work because the DRC technology allows for the introduction of oxygen as a second gas to react with arsenic ions to form AsO, measurable at mass 91. This avoids the interference of chlorine which, with the use of argon gas, causes a polyatomic isobaric spectral overlap at mass 75. Additionally, the system is totally integrated and automated.  For urine samples, the only sample preparation required is dilution with the mobile phase before loading it onto the column, to obtain a composition and pH similar to that of the mobile phase.

Several years ago we introduced a novel method for the measurement of As metabolites in blood. The method is effective provided the total blood As concentration exceeds 5 µg/L.  In this case, blood samples undergosubstantial preparation before they are loaded onto the HPLC column.7, 8 The measurement of As in toenails is relatively new for this laboratory. This biomarker is being employed in an ongoing study of As exposure and child IQ in Maine and New Hampshire. We have also now validated the method for the measurement of manganese in toenails.

Biochemical Analyses

A novel component of the Trace Metals Facility Core is the provision for measurements of metal-binding proteins which are promising as biomarkers of diseases of interest to the CEHNM. While the biology of transferrin and transferrin receptor are reasonably well understood, the physiology and pathophysiology of lactoferrin, lactoferrin receptor and melanotransferrin are only beginning to emerge. Numerous reports demonstrate the potential of the transferrin-family of proteins as biomarkers for the studies of neurodegenerative disease and, possibly, immune-related respiratory disorders, and cancer. 14-21

The Facility Core will offer investigators the opportunity to analyze an array of metal-binding proteins in serum or plasma. With one exception (ferritin), these methods are not routinely available. We measure serum transferrin and ceruloplasmin by RID, transferrin receptors by ELISA and ferritin by RIA. Both plasma and serum lactoferrin are measured using a commercially available immunoassay. Plasma lactoferrin concentrations represent basal levels, while serum concentrations include amounts released by neutrophils.