Liquid chromatography-inductively coupled plasma-based metallomic approaches to probe health-relevant interactions between xenobiotics and mammalian organisms

In mammals, the transport of essential elements from the gastrointestinal tract to organs is orchestrated by biochemical mechanisms which have evolved over millions of years. The subsequent organ-based assembly of sufficient amounts of metalloproteins is a prerequisite to maintain mammalian health and well-being. The chronic exposure of various human populations to environmentally abundant toxic metals/metalloid compounds and/or the deliberate administration of medicinal drugs, however, can adversely affect these processes which may eventually result in disease. A better understanding of the perturbation of these processes has the potential to advance human health, but their visualization poses a major problem. Nonetheless, liquid chromatography-inductively coupled plasma-based 'metallomics' methods, however, can provide much needed insight. Size-exclusion chromatography-inductively coupled plasma atomic emission spectrometry, for example, can be used to visualize changes that toxic metals/medicinal drugs exert at the metalloprotein level when they are added to plasma in vitro. In addition, size-exclusion chromatography-inductively coupled plasma mass spectrometry can be employed to analyze organs from toxic metal/medicinal drug-exposed organisms for metalloproteins to gain insight into the biochemical changes that are associated with their acute or chronic toxicity. The execution of such studies-from the selection of an appropriate model organism to the generation of accurate analytical data-is littered with potential pitfalls that may result in artifacts. Drawing on recent lessons that were learned by two research groups, this tutorial review is intended to provide relevant information with regard to the experimental design and the practical application of these aforementioned metallomics tools in applied health research.     

Biogeographic and Evolutionary Patterns of Trace Element Utilization in Marine Microbial World

Trace elements are required by all organisms, which are key components of many enzymes catalyzing important biological reactions. Many trace element-dependent proteins have been characterized; however, little is known about their occurrence in microbial communities in diverse environments, especially the global marine ecosystem. Moreover, the relationships between trace element utilization and different types of environmental stressors are unclear. In this study, we used metagenomic data from the Global Ocean Sampling expedition project to identify the biogeographic distribution of genes encoding trace element-dependent proteins (for copper, molybdenum, cobalt, nickel, and selenium) in a variety of marine and non-marine aquatic samples. More than 56,000 metalloprotein and selenoprotein genes corresponding to nearly 100 families were predicted, becoming the largest dataset of marine metalloprotein and selenoprotein genes reported to date. In addition, samples with enriched or depleted metalloprotein/selenoprotein genes were identified, suggesting an active or inactive usage of these micronutrients in various sites. Further analysis of interactions among the elements showed significant correlations between some of them, especially those between nickel and selenium/copper. Finally, investigation of the relationships between environmental conditions and metalloprotein/selenoprotein families revealed that many environmental factors might contribute to the evolution of different metalloprotein and/or selenoprotein genes in the marine microbial world. Our data provide new insights into the utilization and biological roles of these trace elements in extant marine microbes, and might also be helpful for the understanding of how these organisms have adapted to their local environments.     

Assessment of trace elements in human brain using inductively coupled plasma mass spectrometry

Recent brain research reveals a major role of trace elements in various diseases such as multiple sclerosis, Alzheimer's and Wilson's disease. The majority of published tissue concentrations dates back decades, and was assessed with various methods. Little is known about hemispherical differences, the correlation of trace elements or age-dependent changes in the human brain. Thus, the aim of this study was to examine trace element concentrations in different human brain regions after whole brain formalin fixation.549 samples of 13 brain regions were investigated in 11 deceased subjects without known history of brain pathology. Regional wet-to-dry mass ratios and concentrations of iron, copper, magnesium, manganese, calcium and zinc were determined using inductively coupled plasma mass spectrometry.Cortical gray matter revealed higher water content (wet-to-dry mass ratios 5.84–6.40) than white matter regions (wet-to-dry mass ratios 2.95–3.05). Element concentrations displayed specific regional differences. Good linear correlation of concentrations between elements was found for iron/copper as well as for manganese/magnesium (Spearman's rank correlation coefficient 0.74 and 0.65, respectively). Significant inter-hemispherical differences were found for copper in occipital white matter, for magnesium and calcium in putamen and for iron and copper in temporal white matter. An age dependent increase was seen in cortical gray matter for calcium, for magnesium in all regions except in cortical gray matter, for copper in substantia nigra and for zinc in occipital cortex.The presented trace element concentrations can serve as a fundamental basis for further brain research. Wet-to-dry mass ratios allow a comparison with reference data from other studies.     

Effects of storage conditions on the stability and distribution of clinical trace elements in whole blood and plasma: Application of ICP-MS

BackgroundKnowledge of trace element stability during sample handling and preservation is a prerequisite to produce reliable test results in clinical trace element analysis.MethodAn alkaline dissolution method has been developed using inductively coupled plasma mass spectrometry to quantify eighteen trace element concentrations: vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, bromine, molybdenum, cadmium, antimony, iodine, mercury, thallium, lead, and bismuth in human blood, using a small sample volume of 0.1 mL. The study evaluated the comparative effects of storage conditions on the stability of nutritionally essential and non-essential elements in human blood and plasma samples stored at three different temperatures (4 °C, −20 °C and −80 °C) over a one-year period, and analysed at multiple time points. The distribution of these elements between whole blood and plasma and their distribution relationships are illustrated using blood samples from 66 adult donors in Queensland.ResultsThe refrigeration and freezing of blood and plasma specimens proved to be suitable storage conditions for many of the trace elements for periods up to six months, with essentially unchanged concentrations. Substantially consistent recoveries were obtained by preserving specimens at −20 °C for up to one year. Ultra-freezing of the specimens at −80 °C did not improve stability; but appeared to result in adsorption and/or precipitation of some elements, accompanied by a longer sample thawing time. A population sample study revealed significant differences between the blood and plasma concentrations of six essential elements and their relationships also varied significantly for different elements.ConclusionBlood and plasma specimens can be reliably stored at 4 °C for six months or kept frozen at −20 °C up to one year to obtain high quality test results of trace elements.     

Biological X-ray absorption spectroscopy (BioXAS): a valuable tool for the study of trace elements in the life sciences

Using X-ray absorption spectroscopy (XAS) the binding modes (type and number of ligands, distances and geometry) and oxidation states of metals and other trace elements in crystalline as well as non-crystalline samples can be revealed. The method may be applied to biological systems as a 'stand-alone' technique, but it is particularly powerful when used alongside other X-ray and spectroscopic techniques and computational approaches. In this review, we highlight how biological XAS is being used in concert with crystallography, spectroscopy and computational chemistry to study metalloproteins in crystals, and report recent applications on relatively rare trace elements utilised by living organisms and metals involved in neurodegenerative diseases.     

Combination of PAGE and LA-ICP-MS as an analytical workflow in metallomics: state of the art, new quantification strategies, advantages and limitations

Metallomics (more specifically, metalloproteomics) is an emerging field that encompasses the role, uptake, transport and storage of trace metals, which are essential to preserve the functions of proteins within a biological system. The current strategies for metal-binding and metalloprotein analysis based on the combination of polyacrylamide gel electrophoresis (PAGE) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) are discussed in this review. The advantages, limitations and the most recently developed and applied quantification approaches for this methodology are also described.     

Trace Element Levels in Scalp Hair of School Children in Shigatse,Tibet, an Endemic Area for Kaschin-Beck Disease (KBD)

Kaschin-Beck disease (KBD) is an endemic osteoarthritis, and the etiology is closely related with levels of trace elements in the human body. Currently, it is clear that the selenium (Se) status of children in KBD areas is lower than that in non-KBD areas in the Tibetan Plateau, whereas role of other elements are yet unknown. This study aimed to assess some essential trace elements (Se, Mo, Mn, Zn, Fe, Cu, Co, and Sr) in children using scalp hair as a biomarker, and 157 samples from school children aged 8–14 years old were collected from both KBD and non-KBD areas in Shigatse, Tibet. Se and Mo were measured by inductive coupled plasma mass spectrometry, and the other elements were determined by inductive coupled plasma optical emission spectrometry. Compared with the non-KBD areas, Se, Mo, Mn, Fe, Zn, Co, and Sr levels of children in KBD areas were found to be significantly different (P < 0.05); while in linear discriminant analysis, only Se and Zn were found to contribute to the KBD prevalence in the study area. The hair Se level of children in KBD areas ranged from 0.115 to 0.299 mg/kg, while in non-KBD areas it ranged from 0.135 to 0.519 mg/kg. The Zn content of children’s hair was between 83 and 207 mg/kg in KBD areas, while it was 37 and 219 mg/kg in non-KBD areas. Lower Se and higher Zn levels in children in KBD areas was found when compared with non-KBD groups. In addition, Mo levels were found to be different between KBD areas and non-KBD areas on the opposite side of the Yarlung Zangbo River, but no close relationship was shown because there was no difference compared with the non-KBD area on the same side of the river. Our observations suggest that Se deficiency is still an important factor for the occurrence and prevalence of KBD, while the relationship between Zn and KBD needs to be further explored in the Tibetan Plateau.     

Trace elements and melanoma.

Melanoma incidence has been steadily increasing in recent years in most western countries, thus suggesting a role of environmental risk factors. Among these determinants, it has been hypothesized that some trace elements of nutritional and toxicological interest may be implicated in the etiology of the disease. We examined patients with newly diagnosed melanoma of the skin and population controls from the Modena province northern Italy. Clinical and dietary data were collected through questionnaires, and toenails were sampled for trace element determination. Levels of cadmium, chromium, lead, selenium, zinc, copper and iron in toenails were measured by inductively coupled plasma optical emission spectrometry and by neutron activation analysis. Data obtained from 58 cases and 58 controls indicated higher levels of copper and lower concentrations of iron in melanoma patients, whilst no other differences were seen for the remaining elements. Patterns of correlations of zinc and copper with the estimated intake of some dietary factors were different between cases and controls. Results of the present study suggest that abnormal intake or metabolism of copper and of iron might be implicated in the etiology of melanoma, whilst they do not indicate an involvement of exposure to cadmium, chromium, lead, selenium and zinc in this disease.     

Insights into metalloproteins and metallodrugs from electron paramagnetic resonance spectroscopy

Metal ions play an important role in diverse biological processes, and much of the basic knowledge derived from studying native bioinorganic systems are applied in the synthesis of new molecules with the aim of diagnosing and treating diseases. At first glance, metalloproteins and metallodrugs are very different systems, but metal ion coordination, redox chemistry and substrate binding play essential roles in advancing both of these research fields. In this article, we discuss recent metalloprotein and metallodrug studies where electron paramagnetic resonance spectroscopy served as a major tool to gain a better understanding of metal-based structures and their function.     

New hope for the diagnosis and therapy of Alzheimer's disease

Improperly folded metal cofactor-containing proteins (e.g., copper chaperone for superoxide dismutase, CCS) are believed to play a key role in several protein-misfolding diseases (e.g., Alzheimer's disease or Amyotrophic Lateral Sclerosis) because under regular physiological conditions, metallochaperones activate or stabilize the native conformation of important metalloproteins (e.g., superoxide dismutase) in certain cellular processes. For an improved diagnosis and therapy of neurodegenerative diseases, new methodologies have to be developed that enable a well-defined differentiation between properly folded and inactive metalloproteins in clinical samples. In the literature it is reported that different high molecular mass metal-containing proteins were isolated in brain samples from Alzheimer's patients and in vegetables by using a 2-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. In the present article, selected results of these studies are scrutinized and compared with some results obtained by a standardized method termed 'quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE)'. Conclusively, QPNC-PAGE is a highly efficient approach used by biochemists to resolve native and denatured metalloproteins (MW 6 - > or = 200 kDa) in complex protein mixtures.     

Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteins contained in rabbit plasma by size-exclusion chromatography–inductively coupled plasma atomic emission spectroscopy

Analytical methods which are capable of determining the plasma or serum metalloproteome have inherent diagnostic value for human diseases associated with increased or decreased concentrations of specific plasma metalloproteins. We have therefore systematically developed a method to rapidly determine the major Cu-, Fe-, and Zn-containing metalloproteins in rabbit plasma (0.5 mL) based on size-exclusion chromatography (SEC; stationary phase Superdex 200, mobile phase phosphate-buffered saline pH 7.4) and the simultaneous online detection of Cu, Fe, and Zn in the column effluent by an inductively coupled plasma atomic emission spectrometer (ICP-AES). Whereas most previous studies reported on the analysis of serum, our investigations clearly demonstrated that the analysis of plasma within 30 min of collection results in the detection of one more Cu peak (blood coagulation factor V) than has been previously reported (transcuprein, ceruloplasmin, albumin-bound Cu, and small molecular weight Cu). The average amount of Cu associated with these five proteins corresponded to 21, 18, 21, 30 and 10% of total plasma Cu, respectively. In contrast, only two Fe metalloproteins (ferritin and transferrin, corresponding to an average of 9 and 91% of total plasma Fe) and approximately five Zn metalloproteins (α₂-macroglobulin and albumin-bound Zn, which corresponded to an average of plasma Zn) were detected. Metalloproteins were assigned on the basis of the coelution of the corresponding metal and protein identified by immunoassays or activity-based enzyme assays. The SEC-ICP-AES approach developed allowed the determination of approximately 12 Cu, Fe, and Zn metalloproteins in rabbit plasma within approximately 24 min and can be applied to analyze human plasma, which is potentially useful for diagnosing Cu-, Fe-, and Zn-related diseases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Parts of the work described in this paper were presented at HPLC 2007 in Ghent, Belgium. An erratum to this article can be found at     

Remarkable effect of mobile phase buffer on the SEC-ICP-AES derived Cu, Fe and Zn-metalloproteome pattern of rabbit blood plasma

The development of an analytical method to quantify the major Cu, Fe and Zn-containing metalloproteins in mammalian plasma has been recently reported. This method is based on the separation of plasma proteins by size exclusion chromatography (SEC) followed by the on-line detection of the metalloproteins by an inductively coupled plasma atomic emission spectrometer (ICP-AES). To assess whether the mobile phase buffer can affect the SEC-ICP-AES-derived metalloproteome pattern, thawed rabbit plasma was analyzed using phosphate buffered saline (PBS)-buffer (0.15 M, pH 7.4), Tris-buffer (0.1 and 0.05 M, pH 7.4), Hepes-buffer (0.1 M, pH 7.4) or Mops-buffer (0.1 M, pH 7.4). In contrast to the Cu-specific chromatograms, the Fe and Zn-specific chromatograms that were obtained with Tris, Hepes and Mops-buffer were considerably different from those attained with PBS-buffer. The Tris, Hepes and Mops-buffer mediated redistribution of ~25% plasma Zn(2+) from <100 kDa to >100-600 kDa plasma proteins and to a smaller extent to a <10 kDa (Tris)(2)Zn(2+)-complex can be rationalized in terms of the abstraction of Zn(2+) from the weak binding site on albumin. In contrast, only Hepes and Mops-buffer redistributed ~20% of plasma Fe(3+) from the <100 kDa to the >600 kDa elution range. Based on these results and considering that the utilization of PBS-buffer has previously resulted in the detection of a number of Cu, Fe and Zn-containing metalloentities in rabbit plasma that was most consistent with literature data, this mobile phase buffer is recommended for metallomic studies regarding mammalian blood plasma.     

Lipid Integration in Neurodegeneration: An Overview of Alzheimer’s Disease

Various types of lipids and their metabolic products associated with the biological membrane play a crucial role in signal transduction, modulation, and activation of receptors and as precursors of bioactive lipid mediators. Dysfunction in the lipid homeostasis in the brain could be a risk factor for the many types of neurodegenerative disorders, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. These neurodegenerative disorders are marked by extensive neuronal apoptosis, gliosis, and alteration in the differentiation, proliferation, and development of neurons. Sphingomyelin, a constituent of plasma membrane, as well as its primary metabolite ceramide acts as a potential lipid second messenger molecule linked with the modulation of various cellular signaling pathways. Excessive production of reactive oxygen species associated with enhanced oxidative stress has been implicated with these molecules and involved in the regulation of a variety of different neurodegenerative and neuroinflammatory disorders. Studies have shown that alterations in the levels of plasma lipid/cholesterol concentration may result to neurodegenerative diseases. Alteration in the levels of inflammatory cytokines and mediators in the brain has also been found to be implicated in the pathophysiology of neurodegenerative diseases. Although several mechanisms involved in neuronal apoptosis have been described, the molecular mechanisms underlying the correlation between lipid metabolism and the neurological deficits are not clearly understood. In the present review, an attempt has been made to provide detailed information about the association of lipids in neurodegeneration especially in Alzheimer’s disease.     

Investigation of thermostable metalloproteins in Perna perna mussels from differentially contaminated areas in Southeastern Brazil by bioanalytical techniques

Metallomic studies regarding environmental contamination by metals are of value in elucidating metal uptake, trafficking, accumulation and metabolism in biological systems. Many proven bioindicator species, such as bivalves, have not yet, however, been well-characterized regarding their metalloprotein expression in response to environmental contaminants. In this context, the aim of the present study was to investigate metalloprotein expressions in the thermostable protein fraction of muscle tissue and digestive glands from mussels (Perna perna) from three differentially metal-contaminated sites in Southeastern Brazil in comparison with a reference site. The thermostable protein fractions were analyzed by SDS-PAGE and SEC-HPLC-ICP-MS. Metal content was also determined in both the crude and the purified extracts. Several inter-organ differences were observed, which is to be expected, while inter-site differences regarding thermostable protein content were also verified, indicating accumulation of these elements in muscle tissue and digestive glands and disruption of homeostasis of essential elements, with detoxification attempts by metal-bound proteins, since all metalloproteins present in both matrices eluted bound to at least one non-essential metal. These results are also noteworthy with regard to the adopted reference site, that also seems to be contaminated by toxic metals.     

Detection of Urine C2C and Trace Element Level in Patients with Knee Osteoarthritis

The objective is to determine the relation between severity of knee osteoarthritis (KOA) and levels of Collagen type II metabolite (C2C) and trace elements in the urine. The urine sample and knee joint films (anteroposterior and lateral) from the KOA patients and control subjects were collected. The KOA patients were divided into five groups (controls and grades I–IV) according to the Kellgren–Lawrence radiographic grading standards. Urine levels of C2C and trace elements were detected by enzyme-linked immunosorbent assay and inductively coupled plasma atomic emission spectrometry, respectively. Urine C2C levels in the KOA subjects (261.235 ± 39.944 pg/ml) were higher than those of the control group (218.341 ± 22.270 pg/ml). The Fe content in KOA groups was significantly lower than that of control group (group IV > group III > group II > group I or controls). The contents of Cu and Zn were also significantly higher in the KOA patients than in the control group (p < 0.05). However, Cr, Al, Cd, Ni, and Se levels of KOA patients were not significantly different from those of the controls (p > 0.05). Determination of the urine levels of C2C and trace elements may prove to be informative for an early diagnosis of KOA. It can also assist in the prognosis judgment of the disease and selecting an appropriate therapeutic regimen.     

Synchrotron radiation for direct analysis of metalloproteins on electrophoresis gels

Metalloproteomics requires analytical techniques able to assess and quantify the inorganic species in metalloproteins. The most widely used methods are hyphenated techniques, based on the coupling of a high resolution chromatographic method with a high sensitivity method for metal analysis in solution. An alternative approach is the use of methods for solid sample analysis, combining metalloprotein separation by gel electrophoresis and direct analysis of the gels. Direct methods are based on beam analysis, such as lasers, ion beams or synchrotron radiation beams. The aim of this review article is to present the main features of synchrotron radiation based methods and their applications for metalloprotein analysis directly on electrophoresis gels. Synchrotron radiation X-ray fluorescence has been successfully employed for sensitive metal identification, and X-ray absorption spectroscopy for metal local structure speciation in proteins. Synchrotron based methods will be compared to ion beam and mass spectrometry for direct analysis of metalloproteins in electrophoresis gels.     

Probing the viral metallome: searching for metalloproteins in bacteriophage λ-- the hunt begins

Although the proteome and genome of bacteriophages are well developed, there is little knowledge about metals and their interactions with the phages, even though metals have been observed in stabilizing phage particles. With expanding studies of phage display and its promising applications, metalloprotein investigations in the bacteriophage areas are necessary to understand whether or not metalloproteins are included in the viral coat proteome. Since these virus studies are still in their infancy, lambda phage was chosen due to its high metal-binding potential as suggested by the cysteine/methionine rich proteins in the viral coat. After large-scale preparation and further purification of lambda phage according to standard protocols, state-of-the-art metallomics techniques via combinations of chromatographies and mass spectrometries were utilized for screening metal-associated species in lambda phage. The lambda phage sample was first separated using non-denaturing size exclusion chromatography with selective metal detection by ICPMS for screening associated metals and generating size distribution fractions for the various metal species, some of which include metalloproteins. Various molecular size distribution patterns were exhibited for the metals detected, Mn, Fe, Co, Ni, Cu and Zn, at different molecular weight ranges. On the other hand numerous other metals were not associated with the coat proteins, as they were not detected in the different molecular weight fractions. Further identification for putative metallopeptides and metalloproteins was accomplished by collecting various metal species' fractions offline and subsequently analyzing tryptically-digested fractions via nanoLC-Chip-ESI-MS. By searching appropriate MS databases with both Spectrum Mill and MASCOT search engines, the main capsid protein, gpE, a capsid decoration protein, gpD, and main tail component protein, gpV, were found and are known for associations with the detected transition metals. These findings will likely provide valuable information for lambda phage engineered applications.     

Human biomonitoring of 73 elements in blood,serum, erythrocytes and urine

BackgroundHuman biomonitoring studies of trace elements in biological fluids are mostly limited to a certain number of elements or biological materials. In this study, we describe the significant extension of a biomonitoring to 73 elements being present in concentration ranges from ng/L to g/L in clinically relevant specimens such as blood, serum, erythrocytes and urine.MethodsThe samples were collected from 102 occupationally non-exposed inhabitants of northern Germany. The elements were determined either by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) in the low concentration range or by inductively coupled plasma optical emission spectrometry (ICP-OES) for essential trace elements and electrolytes.ResultsMean values and selected percentiles of element concentrations are presented for all sample materials. From the results, we calculated the distribution of elements between plasma and blood cells. Application of ICP-MS/MS improves selectivity and accuracy in the determination of elements that are strongly spectrally interfered, such as Cr, Ge, Pd or Ti in blood samples.ConclusionsThis publication provides very valuable information for occupational or environmental hygienists, toxicologists and clinical chemists due to the particularly high number of determined elements and presented concentration ranges.     

Metalloprotein separation and analysis by directly coupled size exclusion high-performance liquid chromatography inductively coupled plasma mass spectroscopy

The feasibility of using directly coupled size exclusion high-performance liquid chromatography inductively coupled plasma mass spectroscopy (HPLC/ICP-MS) for the separation and subsequent elemental analysis of metalloproteins in biological samples has been studied. Data, on up to eight elements, was acquired simultaneously and the reconstructed elemental profiles from the chromatographed samples were quantified by flow injection analysis. Absolute and relative detection limits, reproducibility, operational dynamic range, and linearity of response were initially evaluated by analyzing standards of metallothionein protein of known elemental composition for Cd, Zn, and Cu. There was evidence of displacement of Zn from the protein during chromatography and the substitution of Cu sequestered from the mobile phase. Cd associated with the protein was fully recovered during chromatography. Memory effects, due to protein adsorption to the glassware in the torch box, were minimal and there was no degradation of the resolution of the chromatographed peak during extended transport through the HPLC/ICP-MS interface. The versatility of the technique has been demonstrated by the quantitative multi-element analysis of cytosolic metal-binding proteins separated from the polychaete worm Neanthes arenaceodentata. Fidelity of analysis has been demonstrated by two independent procedures: first, by comparing the elemental profiles obtained by directly aspirating the HPLC eluant into the ICP-MS to those obtained by collecting fractions and quantifying the metal content of the proteins in the conventional analytical mode; second, by comparing the stable isotopic profiles for 114Cd obtained by simultaneous ICP-MS analysis with radiometric profiles of 109Cd obtained by counting radioactivity associated with collected fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zinc status and vacuolar zinc transporters control alkaline phosphatase accumulation and activity in Saccharomyces cerevisiae

Little is known about how metalloproteins in the secretory pathway obtain their metal ion cofactors. We used the Pho8 alkaline phosphatase of the yeast Saccharomyces cerevisiae to probe this process in vivo . We found that both Pho8 activity and protein accumulation are zinc-dependent and decrease in zinc-limited cells. Low Pho8 accumulation was the result of degradation by vacuolar proteases. Surprisingly, the protective effect of zinc on Pho8 stability was not solely due to Zn²⁺ binding to the active-site ligands suggesting that the Pho8 protein is targeted for degradation in zinc-limited cells by another mechanism. Pho8 appears to be a rare example of a metalloprotein whose stability is regulated by its metal cofactor independently of active-site binding. We also assessed which zinc transporters are responsible for supplying zinc to Pho8. We found that the Zrc1 and Cot1 vacuolar zinc transporters play the major role while the Msc2/Zrg17 zinc transporter complex active in the endoplasmic reticulum is not involved. These results demonstrate that the vacuolar zinc transporters, previously implicated in metal detoxification, also deliver zinc to certain metalloproteins within intracellular compartments. These data suggest that Pho8 receives its metal cofactor in the vacuole rather than in earlier compartments of the secretory pathway.