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     

不同灵芝子实体及其粗多糖中微量元素及重金属含量分析*

利用电感耦合等离子发射光谱-质谱联用法(ICP-MS)、原子荧光法(AFS)和原子吸收法(AAS)对不同品种灵芝子实体中的生物必需微量元素、有毒的微量元素及重金属元素的含量进行了测定,并对其营养性和安全性进行了分析。与砷、镉、汞相比,在所有品种的灵芝中重金属铅的含量相对较高,但是在对全国各地收集的灵芝及其培养基分析,灵芝对铅没有生物富集的作用。另外,灵芝粗多糖中各种元素的含量均明显高于提取用的灵芝子实体,其中铅和砷含量高于国家标准,其安全性应该受到重视。     

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5-dithiobis(2-nitrobenzoic acid). Cys¹²², Cys¹²⁴ and Cys¹³² are placed near two catalytic sites, Lys¹⁹⁹ and Lys²⁵², and coordinate a zinc ion, referred to as a proximal zinc ion, and Cys²²³ is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as a distal zinc ion, with His¹³¹ . When the wild-type enzymes C122A (Cys¹²²Ala), C124A (Cys¹²⁴Ala), C132A (Cys¹³²Ala) and C223A (Cys²²³Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys¹²², Cys¹²⁴ and Cys¹³² under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys¹²², Cys¹²⁴ and Cys¹³² was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.     

Levels of Metallic Cations in the Surface Sediments in the Vicinity of the Três Marias Dam Lake (Brazil) Determined by ICP-MS and Microwave Sample Preparation

The presence of toxic elements in sediments is critical in the State of Minas Gerais, which is an important mining area in Brazil. In this work, Ag, As, Ba, Cd, Co, Cr, Cs, Cu, Ga, In, Li, Ni, Pb, Sr, Tl, V and Zn were determined in samples of sediments by ICP-MS after microwave sample preparation. The samples of surface sediments were collected from the vicinity of Três Marias Dam, Brazil. The results using HNO₃+HF+H₃BO₃ and a microwave oven presented adequate precision and accuracy. The accuracy of the method was determined using different certified reference materials. Most of the results agreed within a 95% confidence level. The concentrations of the investigated analytes were below the values of Threshold Effect Level (TEL) and Probable Effect Level (PEL), with the exception of cadmium, chromium, copper, nickel, and zinc. High concentrations of Cr were observed at all the points, Cu and Ni at point SF 011, Cd at point SF 015, and Zn at points SF 011, SF 013, and SF 015. The results demonstrate that the main pollutants were Zn and Cr. Metal mining and processing are potential sources of contamination, since the investigated points are located in areas known for those activities.     

Speciation of trace elements in proteins in human and bovine serum by size exclusion chromatography and inductively coupled plasma-mass spectrometry with a magnetic sector mass spectrometer

 Proteins are separated by size exclusion chromatography while atomic ions from the inorganic elements are detected on-line by inductively coupled plasma-mass spectrometry. A double focusing mass analyzer provides very high sensitivity, low background, and sufficient spectral resolution to separate the atomic ions of interest from most polyatomic ions at the same nominal m/z value. The chromatograms show the distribution of the elements of interest between protein-bound and free fractions and provide the approximate molecular weights of those protein fractions that contain the elements monitored. The distribution of various elements, including V, Mo, Fe, Co, Mn, and lanthanides, in human or bovine serum samples are shown. Alkali metals and Tl are present primarily as free metal ions and are not bound to proteins. Inorganic elements spiked into the serum samples can be followed into various proteins. EDTA does not remove Fe, Pb, Sn, or Th from the proteins but does extract Mn from some proteins. Procedures for determining the effects of breaking disulfide linkages on the metal binding characteristics of proteins are also described. Received: 22 March 1999 / Accepted: 16 June 1999     

Studies on some trace and minor elements in blood

Blood is one of the widely used specimens for biological trace element research because of its biological significance and ease of sampling. We have conducted a study of the blood of the Kalpakkam township population for trace and minor elements. For this purpose, analytical methods have been developed and standardized in our laboratory for the elemental analysis of blood plasma and red cells. Inductively coupled plasma-mass spectrometry (ICP-MS), a relatively new technique, has been applied for the analysis of trace elements. Details regarding spectral interference and matrix interference encountered in the analysis of blood and the methods of correcting them have been discussed. Flame atomic absorption spectrometry (AAS)/atomic emission spectrometry (AES) has been applied for the determination of minor elements. Precision and accuracy of these methods have also been discussed.     

A fast method for multi-metal determination in soil samples by high-resolution inductively-coupled plasma-mass spectrometry (HR–ICP–MS)

Abstract

Methods for multi-elemental quantitative analysis of soil samples by high-resolution inductively-coupled plasma-mass spectrometry (HR–ICP–MS) with preceding one-step wet digestion in a high-pressure microwave system have been developed. The application of aqua regia as a reagent for wet digestion using a high-pressure microwave digestion system, followed by determination using HR–ICP–MS, allows an accurate, simultaneous determination of a large number of heavy metals and metalloids that are considered as potentially hazardous for the environment and health. The values of the concentrations of the elements in the analysed certified reference material (CRM) that have been obtained in this study can be used as indicative values where this CRM is used as a control for the quality of the measurements.     

单方中药马蹄香散剂和几种微生态制剂中氨基酸和微量元素含量的测定和比较

目的 测定马蹄香和微生态制剂中氨基酸和微量元素的含量,深入认识马蹄香和微生态制剂的特点.方法 应用国家标准GB/T 5009.124-2003高效液相法和应用日本岛津公司ICPS-1000Ⅱ型等离子体发射光谱仪测定马蹄香和微生态制剂中氨基酸和微量元素含量.结果 氨基酸含量在乳酸菌素片＞亿活＞马蹄香＞金双歧＞贝飞达＞思连康＞合生原＞妈咪爱.谷氨酸在所有制剂中含量均高,天冬氨酸在多数制剂中含量也高.而胱氨酸在所有制剂中含量均低或未能测出.元素钙、磷、镁和硫含量在马蹄香＞乳酸菌素片＞亿活＞思连康＞金双歧＞妈咪爱＞贝飞达＞合生原.马蹄香中微量元素含量较丰富.结论 马蹄香和不同的微生态制剂所含的氨基酸和微量元素各有特点,这对不同制剂的微量营养素的认识有特殊意义.     

Crystal structure of the carboxyltransferase domain of the oxaloacetate decarboxylase Na+ pump from Vibrio cholerae

Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from oxaloacetate to the prosthetic biotin group. The crystal structure of the carboxyltransferase at 1.7 A resolution shows a dimer of alpha(8)beta(8) barrels with an active site metal ion, identified spectroscopically as Zn(2+), at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn(2+) metal ion, or by Lys178 near the active site, suggesting that Zn(2+) as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from oxaloacetate is discussed.     

A preliminary study of trace elements in plasma protein by gel chromatography combined with SXRF

Fractions of plasma protein of male Kunming mice (body weight 24.2±0.3g), treated with Cisplatin i.p. injection in dose of 10mg/kg, were obtained by separation on Sephadex-G-50 columns, buffered with ammonium acetate to pH 5.7. The SXRF experiments were performed at the BEPC (Beijing Electron Positron Collider) synchrotron radiation facility. The elements (Pt, S, Ca, Fe, Ni, Cu, Zn, Se, Br and Sr) in the fraction of the plasma proteins (< 22KD) were assayed using highly sensitive SXRF. The relative concentrations of elements were calculated by a normalization of Compton scattering intensity around 22 keV, after the normalization for collecting time of X-ray spectrum and the counting of the ion chamber, and subtracting the contribution of the polycarbonate film used for supporting the samples. The determination could prove that the element Pt in plasma was bound with macro-molecularprotein. Cu and S were present in the fraction of the protein in mice treated with Cisplatin and exhibited an increase, the ratio of treated/control were 1.66±0.06 and 1.78±0.33 respectively, whereas Zn decreased to a ratio of 0.78±0.09. Our results are in agreement with others which showed that Cisplatin exposure leads to a marked loss of kidney copper, and a moderate rise in kidney zinc. However, this work mainly focussed on the implementation of this analytical procedure, but not on the results of the investigations of the effect of Cisplatin on trace elements in plasma protein.     

Molybdenum in human whole blood of adult residents of the Merida State (Venezuela)

The concentration of molybdenum was measured in whole blood samples of 418 (244 males and 174 females) apparently normal donors ranging in age from 18 to 27-years old and living in nine different locations in the Mérida State (Venezuela). The geometric mean concentration of molybdenum of 418 subjects was of 2.66+/-0.66 microgL(-1) (range: 1.20-4.80 microgL(-1)). The levels of molybdenum in whole blood samples found in this work were of 2.57+/-0.52 and 2.54+/-0.51 (range: 1.20-4.80 and 1.40-4.20) microgL(-1) for males and females, respectively. The data of the content molybdenum in whole blood had no statistical correlation with age, sex or height above the sea level of the sampling sites. However, there was a tendency to decrease the levels of the element in those sampling sites located in highlands (> or = 1900 m above the sea level). This variability may be due to the source of molybdenum from the soil to the food chain that has affected its levels in donors from these areas under study. The results of this study are compared with values previously reported for subjects studied in other populations.     

不育男性患者和健康男性血液与精浆中微量元素含量的对比分析

目的:研究不育患者精浆和血液中微量元素的含量,为男性不育的诊断和治疗提供理论依据。方法:对73例正常生育组男性和265例男性不育患者的精浆和血液中的锌、铁、铜、钙、镁、镉进行检测分析,分析两组血液和精浆微量元素的差异。结果:不育组患者精浆和血液中锌的含量明显低于正常对照组,铜、镉离子含量明显高于正常对照组,与正常对照组比较均有显著性差异(P<0.01);而两组中的钙、铁、镁的含量接近,差异无统计学意义(P>0.05)。不育组患者精浆中的锌元素水平明显高于血液锌含量,而血液中的镉含量明显高于精浆中的镉含量,差异有明显的统计学意义(P<0.05)。结论:精浆和血液中锌、铜、镉的变化与男性不育密切相关。     

Reliability of the ICP-AES for trace elements studies of biological materials

In order to investigate the reliability of the Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES) for trace element analysis of biological materials, we have carried out extensive investigations on human plasma, using an Applied Research Laboratory’s ICP-AES. When we aspirated the untreated plasma into the spectrometer, we obtained unreliable and nonreproducible results. However, when we pretreated the plasma by wet digestion (to destroy all the organic material), we achieved reproducible and consistent results. It is, therefore, suggested that biological samples should be pretreated, preferably by wet digestion, before being aspirated into the ICP-AES for analysis.