EXPRESS: Landmark Publications in Analytical Atomic Spectrometry: Fundamentals and Instrumentation Development.

The almost-two-centuries history of spectrochemical analysis has generated a body of literature so vast that it has become nearly intractable for experts, much less for those wishing to enter the field. Authoritative, focused reviews help to address this problem but become so granular that the overall directions of the field are lost. This broader perspective can be provided partially by general overviews but then the thinking, experimental details, theoretical underpinnings and instrumental innovations of the original work must be sacrificed. In the present compilation, this dilemma is overcome by assembling the most impactful publications in the area of analytical atomic spectrometry. Each entry was proposed by at least one current expert in the field and supported by a narrative that justifies its inclusion. The entries were then assembled into a coherent sequence and returned to contributors for a round-robin review.

Accurate Quantification and Imaging of Cellular Uptake Using Single-Particle Surface-Enhanced Raman Scattering.

Understanding the uptake, distribution, and stability of gold nanoparticles (NPs) in cells is of fundamental importance in nanoparticle sensors and therapeutic development. Single nanoparticle imaging with surface-enhanced Raman spectroscopy (SERS) measurements in cells is complicated by aggregation-dependent SERS signals, particle inhomogeneity, and limited single-particle brightness. In this work, we assess the single-particle SERS signals of various gold nanoparticle shapes and the role of silica encapsulation on SERS signals to develop a quantitative probe for single-particle level Raman imaging in living cells. We observe that silica-encapsulated gap-enhanced Raman tags (GERTs) provide an optimized probe that can be quantifiable per voxel in SERS maps of cells. This approach is validated by single-particle inductively coupled mass spectrometry (spICP-MS) measurements of NPs in cell lysate post-imaging. spICP-MS also provides a means of measuring the tag stability. This analytical approach can be used not only to quantitatively assess nanoparticle uptake on the cellular level (as in previous digital SERS methods) but also to reliably image the subcellular distribution and to assess the stability of NPs in cells.

Metal enrichment in ice-melt water and uptake by chironomids as possible legacy of World War One in the Italian Alps.

Relics of World War One (WW1) were buried in alpine glaciers around 100 years ago. Today, these are emerging from the ice due to widespread glacier retreat, and are in direct contact with glacial meltwater-fed streams. To address a possible emergent contamination, we quantified major and trace elements (M-TEs) by mass spectrometry in water and larvae of Diamesa zernyi from three glacial streams fed by glaciers differently impacted by the Italian Austro-Hungarian war, in the Adamello-Presanella mountain range (Italian Alps): Lares and Presena, the two main battlefields, and Amola, 8 km from the front. M-TEs in stream water were interpreted using the crustal enrichment factor (EFc) while larval uptake was quantified by adopting the bioaccumulation factor (BAF). Despite low M-TEs concentrations in the water, in a range between 1 ng L-1 (Ag, Ta) and 1-2 mg L-1 (Al, Fe, Mg), low to moderate enrichments (10 ≥ EFc≥ 6) were observed for Sb and U in Presena and for Ag, As, Bi, Cd, Li, Mo, Pb, Sb and U in Lares. In addition, M-TE mass concentrations in larvae were up to ninety thousand times higher than in water, from 20 to 50 ng g-1 dry weight (d.w.; for Bi, Sb, Ta, Tl) to 1-4 mg g-1 d.w. (for Al, Fe, Na, and Mg). Larvae from Lares accumulated the largest amount of metals and metalloids, including those mostly used in the manufacture of artillery shells (As, Cu, Ni, Pb, Sb; BAFs from 375 to about 11,500). This was expected as most of the WW1 battles in this mountain range were fought on the Lares glacier, where the greatest number of war relics are emerging. These results provide preliminary evidence of water contamination and bioaccumulation of metals and metalloids by glacial fauna as a possible legacy of WW1 in the Alps.

Gadolinium during human pregnancy following administration of gadolinium chelate before pregnancy.

Gadolinium (Gd), a toxic rare earth element, has been shown to dissociate from chelating agents and bioaccumulate within tissues, raising concerns about the possibility of their remobilization during pregnancy with subsequent free Gd exposures to developing fetuses. Gd chelates are among the most commonly used magnetic resonance imaging (MRI) contrast agents. This investigation was undertaken after the detection of elevated Gd (800-1000× higher than the usual rare earth element levels) in preliminary unpublished studies from the placentae of subjects in the NIH ECHO/UPSIDE Rochester Cohort Study and unpublished studies from placentae analyzed in formalin-fixed placental specimens from Surgical Pathology at the University of Rochester. Fifteen pregnancies with elevated Gd were studied (12 first pregnancies and 3 second pregnancies). Maternal bloods were collected from all three trimesters, maternal, and cord (fetal) bloods at delivery as well as placental tissue. Breastmilk was also collected from selected mothers. It was determined that Gd was present in maternal bloods from all three trimesters, and in cord bloods and breastmilk in both first and second pregnancies. These results emphasize the need to fully appreciate the implications of pre-pregnancy exposure to Gd chelates and its potential effects on maternal and fetal health.

Comparison of trace element concentrations in paired formalin-fixed paraffin-embedded and frozen human placentae.

INTRODUCTION: There is increasing interest in measuring metals concentrations in human placentas to better understand physiology, disease, and toxic and diagnostic exposures. For these purposes, formalin-fixed paraffin embedded (FFPE) tissues obtained at clinical pathology examination represent a valuable potential store of well-characterized tissues for analysis. However, the limited data that exist comparing metal concentrations in FFPE tissue to recently collected frozen tissues paints a confusing picture, and there is no published data directly comparing frozen and FFPE placental villus tissues. METHODS: Paired samples of fresh frozen and FFPE tissue from 22 rapidly processed human singleton placentae were weighed and digested using standard clean laboratory procedures and subsequently analyzed for a suite of 13 metals using a PerkinElmer DRC II ICP-MS. The analytical results were compared using either a paired t-test or a sign test depending on data normality. RESULTS: Concentrations of metals (aluminum (Al), arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), gadolinium (Gd), mercury (Hg), manganese (Mn), lead (Pb), strontium (Sr), and zinc (Zn)) measured in both types of tissue preparations (frozen and FFPE) displayed a consistent range with other studies and did not display significantly different values from each of the paired specimens for any of the 13 specific metals analyzed. DISCUSSION: Within placentae, metals concentrations of measured trace, toxic and diagnostic elements (Al, As, Ba, Cd, Cr, Cu, Fe, Gd, Hg, Mn, Pb, Sr, and Zn) are consistent between FFPE and fresh placental villus tissue, without indications of systematic element loss or bias. FFPE from archived pathology specimens may offer an important and convenient alternative for measuring trace metals in human frozen placental tissues.

ICP-MS measurements of elemental release from two palladium alloys into a corrosion testing medium for different solution volumes and agitation conditions.

STATEMENT OF PROBLEM: The in vivo release of Pd from palladium alloys into the oral environment and sensitivity reactions by patients has been of concern. However, little information is available about the variation in elemental release from different palladium alloys. PURPOSE: The purpose of this in vitro study was to compare the elemental release into a corrosion-testing medium from a high-palladium alloy (Freedom Plus, 78Pd-8Cu-5Ga-6In-2Au) and a Pd-Ag alloy (Super Star, 60Pd-28Ag-6In-5Sn) under different conditions. MATERIAL AND METHODS: Alloys were cast into Ø12×1-mm-thick disks, subjected to simulated porcelain-firing heat treatment, polished, and ultrasonically cleaned in ethanol. Three specimens of each alloy were immersed for 700 hours in a solution for in vitro corrosion testing (ISO Standard 10271) that was maintained at 37 °C. Two solution volumes (125 mL and 250 mL) were used, and the solutions were subjected to either no agitation or agitation. Elemental compositions of the solutions were analyzed by using inductively coupled plasma-mass spectroscopy (ICP-MS). Concentrations of released elements from each alloy for the 2 solution volumes and agitation conditions were compared by using the restricted maximum likelihood estimation method with a 4-way repeated-measures ANOVA, the Satterwhite degrees of freedom method, a lognormal response distribution, and the covariance structure of compound symmetry. RESULTS: For the 4 combinations of solution volume and agitation conditions, the mean amount of palladium released was 3 orders of magnitude less for the Pd-Ag alloy (0.009 to 0.017 µg/cm2 of alloy surface) compared with the Pd-Cu-Ga alloy (17.9 to 28.7 µg/cm2). Larger mean amounts of Sn, Ga, Ag, and In (0.29 to 0.39, 0.57 to 0.83, 0.71 to 1.08, and 0.91 to 1.25 µg/cm2, respectively) compared with Pd were released from the Pd-Ag alloy. Smaller amounts of Cu, Ga, and In (4.8 to 9.9, 5.9 to 12.8, and 4.2 to 9.5 µg/cm2, respectively) compared with Pd were released from the Pd-Cu-Ga alloy. The Ru released was much lower for the Pd-Ag alloy (0.002 µg/cm2) than the Pd-Cu-Ga alloy (0.032 to 0.053 µg/cm2). Statistically significant differences (P<.001) in elemental release were found for the factors of alloy and element and the alloy×element interaction. Significant differences were found for the solution volume (P=.022), solution volume×element interaction (P=.022), and alloy×solution volume×element interaction (P=.004). No significant effect was found for agitation condition. CONCLUSIONS: The relative amounts of released elements from each alloy were not proportional to the relative amounts in the composition. The amounts of Pd and Ga released from the Pd-Cu-Ga alloy were consistent with the breakdown of a Pd2Ga microstructural phase and perhaps some dissolution of the palladium solid solution matrix. Precipitates, rather than the palladium solid solution matrix, appeared to undergo greater dissolution in the Pd-Ag alloy. The Pd-Ag alloy should have lower risk of adverse biological reactions than the Pd-Cu-Ga alloy.

Dissipation of transmembrane potassium gradient is the main cause of cerebral ischemia-induced depolarization in astrocytes and neurons.

Membrane potential (VM) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K+ and glutamate has been shown to underlie an ischemia-induced VM depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K+ gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K+ gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K+ contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K+ loss and depolarization should facilitate the development of future stroke therapy.

Trace Element Analysis of Borrelia burgdorferi by Inductively Coupled Plasma-Sector Field Mass Spectrometry.

Transition metal ions play important structural, regulatory, and catalytic roles in all biological systems by serving as cofactors for proteins. Due to their relatively low levels in the cell compared to abundant metal ions such as potassium and magnesium, transition metals are often considered micronutrients and referred to as trace elements. Manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are the most prevalent transition metals in the Lyme disease spirochete Borrelia burgdorferi. Here, we describe a method for the accurate measurement of these trace elements in B. burgdorferi utilizing inductively coupled plasma-sector field mass spectrometry (ICP-SFMS).

BosR Is A Novel Fur Family Member Responsive to Copper and Regulating Copper Homeostasis in Borrelia burgdorferi.

The ferric uptake regulator (Fur) family of DNA-binding proteins represses and/or activates gene transcription via divalent metal ion-dependent signal sensing. The Borrelia burgdorferi Fur homologue, also known as Borrelia oxidative stress regulator (BosR), promotes spirochetal adaptation to the mammalian host by directly repressing the lipoproteins required for tick colonization and indirectly activating those required for establishing infection in the mammal. Here, we examined whether the DNA-binding activity of BosR was regulated by any of the four most prevalent transition metal ions in B. burgdorferi, Mn, Fe, Cu, and Zn. Our data indicated that in addition to a structural site occupied by Zn(II), BosR had two regulatory sites that could be occupied by Zn(II), Fe(II), or Cu(II) but not by Mn(II). While Fe(II) had no effect, Cu(II) and Zn(II) had a dose-dependent inhibitory effect on the BosR DNA-binding activity. Competition experiments indicated that Cu(II) had a higher affinity for BosR than Zn(II) or Fe(II). A BosR deficiency in B. burgdorferi resulted in a significant increase in the Cu level but no significant change in the levels of Mn, Fe, or Zn. These data suggest that Cu regulates BosR activity, and BosR in turn regulates Cu homeostasis in B. burgdorferi While this regulatory paradigm is characteristic of the Fur family, BosR is the first one shown to be responsive to Cu(II), which may be an adaptation to the potentially high level of Cu present in the Lyme disease spirochete.IMPORTANCE Transition metal ions serve an essential role in the metabolism of all living organisms. Members of the ferric uptake regulator (Fur) family play critical roles in regulating the cellular homeostasis of transition metals in diverse bacteria, and their DNA-binding activity is often regulated by coordination of the cognate divalent metal ions. To date, regulators with metal ion specificity to Fe(II), Mn(II), Zn(II), and Ni(II) have all been described. In this study, we demonstrate that BosR, the sole Fur homologue in Borrelia burgdorferi, is responsive to Cu(II) and regulates Cu homeostasis in this bacterium, which may be an adaption to potentially Cu-rich milieu in the Lyme disease spirochete. This study has expanded the repertoire of the Fur family's metal ion specificity.

Single Particle ICP-MS: Advances toward routine analysis of nanomaterials.

From its early beginnings in characterizing aerosol particles to its recent applications for investigating natural waters and waste streams, single particle inductively coupled plasma-mass spectrometry (spICP-MS) has proven to be a powerful technique for the detection and characterization of aqueous dispersions of metal-containing nanomaterials. Combining the high-throughput of an ensemble technique with the specificity of a single particle counting technique and the elemental specificity of ICP-MS, spICP-MS is capable of rapidly providing researchers with information pertaining to size, size distribution, particle number concentration, and major elemental composition with minimal sample perturbation. Recently, advances in data acquisition, signal processing, and the implementation of alternative mass analyzers (e.g., time-of-flight) has resulted in a wider breadth of particle analyses and made significant progress toward overcoming many of the challenges in the quantitative analysis of nanoparticles. This review provides an overview of spICP-MS development from a niche technique to application for routine analysis, a discussion of the key issues for quantitative analysis, and examples of its further advancement for analysis of increasingly complex environmental and biological samples. Graphical Abstract Single particle ICP-MS workflow for the analysis of suspended nanoparticles.

Uptake of bright fluorophore core-silica shell nanoparticles by biological systems.

Nanoparticles are used in a variety of consumer applications. Silica nanoparticles in particular are common, including as a component of foods. There are concerns that ingested nano-silica particles can cross the intestinal epithelium, enter the circulation, and accumulate in tissues and organs. Thus, tracking these particles is of interest, and fluorescence spectroscopic methods are well-suited for this purpose. However, nanosilica is not fluorescent. In this article, we focus on core-silica shell nanoparticles, using fluorescent Rhodamine 6G, Rhodamine 800, or CdSe/CdS/ZnS quantum dots as the core. These stable fluorophore/silica nanoparticles had surface characteristics similar to those of commercial silica particles. Thus, they were used as model particles to examine internalization by cultured cells, including an epithelial cell line relevant to the gastrointestinal tract. Finally, these particles were administered to mice by gavage, and their presence in various organs, including stomach, small intestine, cecum, colon, kidney, lung, brain, and spleen, was examined. By combining confocal fluorescence microscopy with inductively coupled plasma mass spectrometry, the presence of nanoparticles, rather than their dissolved form, was established in liver tissues.

Quantitative susceptibility mapping (QSM) of white matter multiple sclerosis lesions: Interpreting positive susceptibility and the presence of iron.

PURPOSE: Within multiple sclerosis (MS) lesions iron is present in chronically activated microglia. Thus, iron detection with MRI might provide a biomarker for chronic inflammation within lesions. Here, we examine contributions of iron and myelin to magnetic susceptibility of lesions on quantitative susceptibility mapping (QSM). METHODS: Fixed MS brain tissue was assessed with MRI including gradient echo data, which was processed to generate field (phase), R2* and QSM. Five lesions were sectioned and evaluated by immunohistochemistry for presence of myelin, iron and microglia/macrophages. Two of the lesions had an elemental analysis for iron concentration mapping, and their phospholipid content was estimated from the difference in the iron and QSM data. RESULTS: Three of the five lesions had substantial iron deposition that was associated with microglia and positive susceptibility values. For the two lesions with elemental analysis, the QSM derived phospholipid content maps were consistent with myelin labeled histology. CONCLUSION: Positive susceptibility values with respect to water indicate the presence of iron in MS lesions, although both demyelination and iron deposition contribute to QSM.

A novel iron- and copper-binding protein in the Lyme disease spirochaete.

Iron and copper are transition metals that can be toxic to cells due to their abilities to react with peroxide to generate hydroxyl radical. Ferritins and metallothioneins are known to sequester intracellular iron and copper respectively. The Lyme disease pathogen Borrelia burgdorferi does not require iron, but its genome encodes a ferritin-like Dps (DNA-binding protein from starved bacteria) molecule, which has been shown to be important for the spirochaete's persistence in the tick and subsequent transmission to a new host. Here, we show that the carboxyl-terminal cysteine-rich (CCR) domain of this protein functions as a copper-binding metallothionein. This novel fusion between Dps and metallothionein is unique to and conserved in all Borrelia species. We term this molecule BicA for Borrelia iron- and copper-binding protein A. An isogenic mutant lacking BicA had significantly reduced levels of iron and copper and was more sensitive to iron and copper toxicity than its parental strain. Supplementation of the medium with iron or copper rendered the spirochaete more susceptible to peroxide killing. These data suggest that an important function of BicA is to detoxify excess iron and copper the spirochaete may encounter during its natural life cycle through a tick vector and a vertebrate host.

Impaired myocardial perfusion reserve and fibrosis in Friedreich ataxia: a mitochondrial cardiomyopathy with metabolic syndrome.

AIMS: Cardiomyopathy produces significant mortality in patients with Friedreich ataxia (FA), a genetic disorder that produces intra-mitochondrial iron accumulation. We sought to test the hypothesis that abnormal myocardial perfusion reserve and fibrosis represent early manifestations of cardiomyopathy. METHODS AND RESULTS: Twenty-six patients with genetically proven FA ages 36 ± 12 years without cardiomyopathy and eight controls underwent cardiac magnetic resonance with adenosine. Precontrast imaging for myocardial iron estimation was performed. Myocardial perfusion reserve index (MPRI) was quantified using the normalized upslope of myocardial enhancement during vasodilator stress vs. rest. Left ventricular (LV) mass and volumes were computed from short-axis cine images. Serologies included lipids, and platelets were isolated for iron quantification using inductively coupled plasma mass spectrometry. Left ventricular ejection fraction and mass averaged 64.1 ± 8.3% and 62.7 ± 16.7 g/m², respectively, indicating preserved systolic function and absence of significant hypertrophy. Myocardial perfusion reserve index quantification revealed significantly lower endocardial-to-epicardial perfusion reserve in patients vs. controls (0.80 ± 0.18 vs. 1.22 ± 0.36, P = 0.01). Lower MPRI was predicted by increased number of metabolic syndrome (met-S) features (P < 0.01). Worse concentric remodelling occurred with increased GAA repeat length (r = 0.64, P < 0.001). Peripheral platelet iron measurement showed no distinction between patients and controls (5.4 ± 8.5 × 10⁻7 vs. 5.5 ± 2.9 × 10⁻7 ng/platelet, P = 0.88), nor did myocardial T2* measures. CONCLUSIONS: Patients with FA have abnormal myocardial perfusion reserve that parallels met-S severity. Impaired perfusion reserve and fibrosis occur in the absence of significant hypertrophy and prior to clinical heart failure, providing potential therapeutic targets for stage B cardiomyopathy in FA and related myocardial diseases.

In vivo atherosclerotic plaque characterization using magnetic susceptibility distinguishes symptom-producing plaques.

OBJECTIVES: We investigated the role of iron deposition in atherosclerotic plaque instability using a novel approach of in vivo plaque characterization by a noninvasive, noncontrast magnetic resonance-based T2* measurement. This approach was validated using ex vivo plaque analyses to establish that T2* accurately reflects intraplaque iron composition. BACKGROUND: Iron catalyzes free radical production, a key step for lipid peroxidation and atherosclerosis development. The parameter T2* measures tissue magnetic susceptibility, which historically has been used to quantify hepatic and myocardial iron. The T2* measurement has not been used for in vivo plaque characterization in patients with atherosclerosis. METHODS: Thirty-nine patients referred for carotid endarterectomy were prospectively enrolled to undergo preoperative carotid magnetic resonance imaging (MRI) and postoperative analysis of the explanted plaque. Clinical history of any symptoms attributable to each carotid lesion was recorded. We could not complete MRI in 4 subjects because of their claustrophobia, and 3 patients scanned before the institution of a neck stabilizer had motion artifact, precluding quantification. RESULTS: Symptomatic patients had significantly lower plaque T2* values (20.0 +/- 1.8 ms) compared with asymptomatic patients (34.4 +/- 2.7 ms, p < 0.001). Analytical methods demonstrated similar total iron (138.6 +/- 36.5 microg/g vs. 165.8 +/- 48.3 microg/g, p = NS) but less low molecular weight Fe(III) (7.3 +/- 3.8 microg/g vs. 17.7 +/- 4.0 microg/g, p < 0.05) in the explanted plaques of symptomatic versus asymptomatic patients, respectively, which is consistent with a shift in iron from Fe(III) to greater amounts of T2*-shortening forms of iron. Mass spectroscopy also showed significantly lower calcium (37.5 +/- 10.8 mg/g vs. 123.6 +/- 19.3 mg/g, p < 0.01) and greater copper (3.2 +/- 0.5 microg/g vs. 1.7 +/- 0.1 microg/g, p < 0.01) in plaques from symptomatic patients. CONCLUSIONS: In vivo measurement of intraplaque T2* using MRI is feasible and distinguishes symptom-producing from non-symptom-producing plaques in patients with carotid artery atherosclerosis. Symptom-producing plaques demonstrated characteristic changes in iron forms by ex vivo analysis, supporting the dynamic presence of iron in the microenvironment of atherosclerotic plaque.

Inductively coupled plasma-mass spectroscopy measurements of elemental release from 2 high-palladium dental casting alloys into a corrosion testing medium.

STATEMENT OF PROBLEM: The biocompatibility of high-palladium alloy restorations has been of some concern due to the release of palladium into the oral environment and sensitivity reactions in patients. PURPOSE: This study measured the in vitro elemental release from a Pd-Cu-Ga alloy and a Pd-Ga alloy into a corrosion testing medium. MATERIAL AND METHODS: Both alloys were cast into 12-mm-diameter x 1-mm-thick disks, subjected to heat treatment that simulated porcelain firing cycles, polished to a 0.05-mm surface finish, and ultrasonically cleaned in ethanol. Two specimens of each alloy were immersed 3 times (at 7, 70, and 700 hours) in an aqueous lactic acid/NaCl solution used for in vitro corrosion testing and maintained at 37 degrees C. The specimens were removed after each immersion time, and the elemental compositions of the solutions were analyzed with inductively coupled plasma-mass spectroscopy (ICP-MS). Elemental concentrations for the 2 alloys at each immersion time were compared with Student t test (alpha=.05). RESULTS: No significant differences in palladium release were found for the 7- and 70-hour solutions, but significant differences were found for the 700-hour solutions. Mean concentrations of palladium and gallium in the 700-hour solutions, expressed as mass per unit area of alloy surface, were 97 (Pd) and 46 (Ga) microg/cm(2) for the Pd-Cu-Ga alloy and 5 (Pd) and 18 (Ga) microg/cm(2) for the Pd-Ga alloy. CONCLUSION: Relative proportions of the elements in the solutions were consistent with the release of palladium and breakdown of microstructural phases found in the alloys. The results suggest that there may be a lower risk of adverse biological reactions with the Pd-Ga alloy than with the Pd-Cu-Ga alloy tested.