On-line reversed-phase liquid chromatography hydride generation emission spectrometry: speciation of arsenic in urine of patients intravenously treated with As2O3

Hydride generation inductively coupled plasma–atomic emission spectrometry (HG ICP–AES) was used as a continuous detection system for the determination of arsenic in the eluate from a high-performance liquid chromatographic (HPLC) system. Four arsenic species [arsenite As(III), arsenate As(V), monomethylarsonate (MMA), and dimethylarsinate (DMA)] present in the urine samples of patients treated intravenously with arsenite, were analyzed separately by HPLC–HG-ICP–AES using a non-polar C₁₈ column. This analytical method allowed the sensitive determination of the arsenic species in the submicrogram per liter range. Urine samples collected on different days after arsenite administration were found to contain arsenite predominantly – monomethylarsonate and dimethylarsinate were also detected.     

Determination of urinary arsenic, mercury, and selenium in steel production workers

The fumes and dust of trace elements and their compounds are very toxic and have been related to an increase in the incidence of diseases. Occupational exposure to toxic metals and metalloids can be determined by means of workplace air measurements and biological monitoring. The aim of our investigation was to determine the concentrations of As, Hg, and Se in urine samples under routine clinical laboratory conditions. To assess the reliability of these methods, critical factors such as detection limit(s), calibration range(s), cost, accuracy, and precision were studied. The method was employed for the quantitative determination of arsenic, mercury, and selenium in urine samples from steel production and quality control workers and healthy unexposed controls. After pretreatment with acids, the samples were digested by means of a microwave oven. Arsenic was determined by hydride atomic absorption spectrometry and mercury was determined by cold vapor atomic absorption spectrometry, whereas selenium was determined by a graphite furnace atomic absorption spectrometry. The results indicate those urinary arsenic, mercury, and selenium levels of the exposed workers are significantly higher than those of the controls. The possibility that these elements are involved in the etiology of diseases is discussed and recommendations are made to improve workplace ventilation and industrial hygiene practices.     

A simple and fast detection technique for arsenic speciation based on high‐efficiency photooxidation and gas‐phase chemiluminescence detection

High‐efficiency photooxidation (HEPO) and gas phase chemiluminescence detection (CL) combined with high‐performance liquid chromatography (HPLC) and hydride generation were developed for speciation of As(III), As(V), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). After chromatography separation, the arsenic species were passed through HEPO which performed efficient photooxidation and converted MMA and DMA to As(V) in several seconds. Then the reaction of ozone and arsine upon hydride generation produced a CL signal as the analytical parameter. The total analytical process was completed within 10 min. The effects of operational parameters such as the concentrations of hydrochloric acid and NaBH₄ solution, carrier gas flow and air gas flow for ozone generation were investigated. Detection limits were 3.7, 10.3, 10.2 and 10.0 µg/L for As(III), As(V), MMA and DMA, respectively. The recoveries of the four arsenic species in human urine sample ranged from 87 to 94%. Copyright © 2009 John Wiley & Sons, Ltd.     

Species variations in the biliary and urinary excretion of arsenate, arsenite and their metabolites

In most mammalian species, inorganic arsenicals are extensively biotransformed and excreted both in unchanged form and as metabolites. In the bile of rats receiving arsenate (AsV) or arsenite (AsIII) we have identified monomethylarsonous acid (MMAsIII), purportedly the most toxic metabolite of inorganic arsenic. As rats are not commonly accepted for studying arsenic metabolism, we carried out a comparative investigation on the excretion of AsV, AsIII and their metabolites in five animal species in order to determine whether they also form MMAsIII from AsV and AsIII. Anaesthetised bile duct-cannulated rats, mice, hamsters, rabbits, and guinea pigs were injected with AsV or AsIII (50 micromol/kg, i.v.) and their bile and urine was collected for 2 h. Arsenic in bile and urine was speciated by HPLC-hydride generation-atomic fluorescence spectrometry and the excretion rates of AsV, AsIII, monomethylarsonic acid (MMAsV), MMAsIII and dimethylarsinic acid (DMAsV) were quantified. All species injected with AsV excreted arsenic preferentially into urine, whereas all animals receiving AsIII, except rabbits, delivered more arsenic into bile than urine. Bile contained almost exclusively trivalent arsenic (i.e. AsIII and/or MMAsIII), whereas AsV, AsIII and DMAsV appeared in urine. Except for guinea pigs, which do not methylate arsenic, the other species formed MMAsIII and excreted it into bile. Having excreted as much as 8% of the dose of AsIII or AsV in 2 h as MMAsIII, rats were by far the most efficient producers of this supertoxic metabolite. Thus, although the rat is not a good model for studying long-term arsenic disposition, this species appears especially valuable in studies on AsIII methyltransferase and in vivo formation of MMAsIII.     

Microplate screening assay for the detection of arsenite-oxidizing and arsenate-reducing bacteria

An efficient, inexpensive microplate colorimetric assay for screening of bacteria which can be used in bioremediation of arsenic was developed. The assay is based on the colorimetric analysis of the precipitates formed upon reaction of silver nitrate with arsenic. The method proved reliable and sensitive for the detection of As[III] oxidizers and As[V] reducers and can be used over a large pH range (5.8-8.4). Seventy-eight bacterial strains isolated from different environments were tested by this method. It also showed agreement with results obtained by high-performance liquid chromatography coupled with inductively coupled plasma atomic emission spectrometry.     

Determination of trivalent methylated arsenicals in rat urine by liquid chromatography-inductively coupled plasma mass spectrometry after solvent extraction

A method for the determination of trivalent arsenicals in urine was examined. Trivalent arsenicals, extracted as complexes with diethylammonium diethyldithiocarbamate (DDDC) into carbon tetrachloride, were determined by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). The trivalent methylated arsenicals monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), and trimethylarsine (TMA) were detected in urine of rats that had received dimethylarsinic acid (DMA(V)) or monomethylarsonic acid (MMA(V)) at concentration of 200 microg ml(-1) in drinking water for 24 weeks. This method is the first to permit quantification of trivalent methylated arsenicals in urine without significant changes in concentration during storage or pretreatment.     

Generation of thioarsenicals is dependent on the enterohepatic circulation in rats

Three minor sulfur-containing arsenic metabolites: monomethylmonothioarsonic acid (MMMTA(V)), dimethylmonothioarsinic acid (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) were recently found in human and animal urine after exposure to inorganic arsenic. However, it remains unclear how the thioarsenicals are formed in the body and then excreted into the urine. It is hypothesized that the generation of thioarsenicals occurs during enterohepatic circulation. To address this hypothesis, male Sprague Dawley (SD) rats and Eisai hyperbilirubinuric (EHB) rats (with deficiency of multidrug resistance-associated protein 2) were orally administered a single dose of inorganic arsenite (iAs(III)) at 3.0 mg kg(-1) of body weight. Five hours after dosing, less than 1.0% of the dose was recovered in the bile of EHB rats, while more than 27% of the dose was recovered in the bile of SD rats, with the majority being monomethylarsinodiglutathione [MMA(SG)(2)] with a small amount of arsenic triglutathione [iAs(SG)(3)]. During the early time periods (3 h and 6 h) the arsenic levels in the liver, red blood cells (RBCs) and plasma of EHB rats were higher than those of SD rats, and approximately 76% and 87% of the dose was recovered in the RBCs of SD and EHB rats, respectively, at day 5 after dosing. However, there were no significant differences in arsenic concentration in urine between the two types of animal. Regarding the arsenic species in the urine of both types of rat, significant levels of thiolated arsenicals MMMTA(V) and DMMTA(V) were detected in SD rat urine, however in EHB rat urine only low levels of DMMTA(V) were detected. The present result of the metabolic balance and speciation study suggests that the formation of MMMTA(V) and DMMTA(V) in rats is dependent on enterohepatic circulation. In addition, in vitro experiments indicated that arsenicals excreted from bile may be transformed by gastrointestinal microbiota into MMMTA(V) and DMMTA(V), which are then absorbed into the bloodstream and finally excreted into the urine.     

Exceptions in patterns of arsenic compounds in urine of acute promyelocytic leukaemia patients treated with As<Subscript>2</Subscript>O<Subscript>3</Subscript>

Arsenic trioxide (As(III) in solution) has been shown to be the most active single agent in combating acute promyelocytic leukemia (APL). It is metabolized and excreted via urine as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V), along with excess As(III). In our study eight APL patients were treated (intravenously) with 0.15 mg As₂O₃/kg/day. During the therapy As(III) and its metabolites were followed in pre- and post-infusion urine using HPLC for separation followed by on-line detection using hydride generation-atomic fluorescence spectrometry. Five patients had a normal excretion pattern of residual arsenic compounds in morning pre-infusion urine, with 15–25 % of As(III), 35–55 % of DMA, 25–30 % of MMA and 1–5 % of As(V), while three patients showed unexpected exceptions from typical excretion patterns of arsenic compounds (i) a high DMA/MMA ratio (factor 5.3), (ii) severe As(III) oxidation (10.2 % As(III) converted to As(V)) or (iii) the presence of an excessive amount of As(III) (average 30.4 % of total arsenic). Intriguing was the occurrence of post-infusion oxidation of As(III) to As(V) observed in almost all patients and being especially high (>40 %) in patient with increased residual As(V). Results indicate that arsenic metabolites patterns can be unpredictable. Observed high levels of un-metabolised As(III) are a warning signal for side effects and for routine determination of arsenic metabolites during first days of treatment. High or low percentages of MMA or DMA did not show any observable effect on treatment results, while clear presence of post-infusion As(V) supports theoretical claims of in vivo oxidation (detoxification) of As(III) to As(V) associated with various metabolic processes.     

The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry

We have developed a method for measuring dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO) in biological samples using gas chromatography with mass spectrometric detection. DMA, TMA, and TMAO were extracted from biological samples into acid after internal standards (labeled with stable isotopes) were added. p-Toluenesulfonyl chloride was used to form the tosylamide derivative of DMA. 2,2,2-Trichloroethyl chloroformate was used to form the carbamate derivative of TMA. TMAO was reduced with titanium(III) chloride to form TMA, which was then analyzed. The derivatives were chromatographed using capillary gas chromatography and were detected and quantitated using electron ionization mass spectrometry (GC/MS). Derivative yield, reproducibility, linearity, and sensitivity of the assay are described. The amounts of DMA, TMA, and TMAO in blood, urine, liver, and kidney from rats and humans, as well as in muscle from fishes, were determined. We also report the use of this method in a pilot study characterizing dimethylamine appearance and disappearance from blood in five human subjects after ingesting [13C]dimethylamine (0.5 mumol/kg body wt). The method we describe was much more reproducible than existing gas chromatographic methods and it had equivalent sensitivity (detected 1 pmol). The derivatized amines were much more stable and less likely to be lost as gases when samples were stored. Because we used GC/MS, it was possible to use stable isotopic labels in studies of methylamine metabolism in humans.     

Arsenic species excretion after controlled seafood consumption

Influence of controlled consumption of marine fish on the urinary excretion of arsenite, arsenate, dimethylarsinic and monomethylarsonic acid (DMA, MMA) was investigated in two experiments. Arsenic species were separated by anion-exchange chromatography and detected with hydride-technique atomic absorption spectrometry (detection limit 1, 10, 2, 2 microg/l). Firstly, 13 probands ate different types of seafood after having refrained from any seafood for 1 week. DMA levels rose from 3.4+/-1.3 microg/g creatinine (n=12; a day before seafood) to a mean peak level of 28.2+/-20.6 microg/g (n=13; 10-23 h after; P<0.001; max. 77.7 microg/g). No other species were excreted before the meal, but small amounts of arsenite (8.5% positive; max. 1.7 microg/g) and MMA (1.2%; 1.6 microg/g) within 2 days after it (n=82). Consumption of white herring caused the highest DMA levels. Secondly, eight probands ingested white herring (dose 3.5 g/kg; DMA content 32.1+/-15.3 ng/g wet weight; n=36). No arsenite, arsenate and MMA was found in the urine or in the herring tissues. The mean DMA mass excreted after the meal (65.3+/-22.0 microg/24 h) was about 6-fold higher than the sum of base DMA excretion (3.0+/-1.7 microg/24 h) and the ingested DMA mass (7.9+/-2.7 microg). This indicates that the elevated DMA excretion after herring consumption is not caused by the metabolism of inorganic arsenic but of other arsenic species present in the fish tissue, e.g. arsenobetaine or fat-soluble arsenic species.     

The influence of chemical form and concentration of arsenic on rice growth and tissue arsenic concentration

Arsenic absorption by rice (Oryza sativa, L.) in relation to the chemical form and concentration of arsenic added in nutrient solution was examined. A 4 × 3 × 2 factorial experiment was conducted with treatments consisting of four arsenic chemical forms [arsenite, As(III); arsenate, As(V); monomethyl arsenic acid, MMAA; and dimethyl arsenic acid, DMAA], three arsenic concentrations [0.05, 0.2, and 0.8 mg As L^-1], and two cultivars [Lemont and Mercury] with a different degree of susceptibility to straighthead, a physiological disease attributed to arsenic toxicity. Two controls, one for each cultivar, were also included. Arsenic phytoavailability and phytotoxicity are determined primarily by the arsenic chemical form present. Application of DMAA increased total dry matter production. While application of As(V) did not affect plant growth, both As(III) and MMAA were phytotoxic to rice. Availability of arsenic to rice followed the trend: DMAA<As(V)<MMAA<As(III). Upon absorption, DMAA was readily translocated to the shoot. Arsenic(III), As(V), and MMAA accumulated in the roots. With increased arsenic application rates the arsenic shoot/root concentration decreased for the As(III) and As(V) treatments. Monomethyl arsenic acid (MMAA), however, was translocated to the shoot upon increased application. The observed differential absorption and translocation of arsenic chemical forms by rice is possibly responsible for the straighthead disorder attributed to arsenic.     

Effects of arsenic exposure among semiconductor workers: a cautionary note on urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine

Arsenic is a notorious environmental toxicant and was found to cause oxidative stress in cultured cells and animals. However, little work has been done in human studies, especially for the population occupationally exposed to arsenic. In order to investigate the effect of occupational exposure to arsenic in oxidative stress, we measured urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) from 90 semiconductor workers including 50 exposed and 40 nonexposed subjects. A highly sensitive and specific isotope dilution LC-MS/MS method was used for quantification of 8-oxodGuo. The levels of inorganic arsenic (iAs3+, iAs5+), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by high-performance liquid chromatography-flow injection atomic absorption spectrometry (HPLC-FIAAS). Results showed that the mean urinary concentrations of total arsenic and 8-oxodGuo were significantly higher for exposed workers compared with the nonexposed workers. In addition, elevated urinary 8-oxodGuo concentrations of exposed workers were correlated with urinary levels of MMA (r = 0.44, P < 0.005) and the extent of primary methylation (the ratio of MMA to inorganic arsenic) (r = 0.40, P < 0.005). These findings suggested that occupational exposure to arsenic could result in the induction of oxidative stress. The presence and/or formation of MMA could play an important role in arsenic-involved injuries.     

Correlation Between Arsenic Concentration in Fish and Human Scalp Hair of People Living in Arsenic-Contaminated and Noncontaminated Areas of Pakistan

The effects of arsenic (As) toxicity due to frequent consumption of arsenic-contaminated fish was estimated by the analysis of scalp hair of adult males, living near arsenic-contaminated area of Pakistan. For comparison purposes, scalp hair samples were also collected from the inhabitants of Hyderabad city consuming fish species with low levels of As, collected from Indus River. Concentration of As in scalp hair samples was analyzed by using hydride generation atomic absorption spectrometry (HG AAS), after microwave-assisted acid digestion. The accuracy of the As measurement was tested simultaneously analyzing certified reference material. The concentration of As in muscle tissues of fish species were found in the range of 2.11 to 14.1?μg/g and 1.92 to 12.2?μg/g, collected from arsenic-contaminated and noncontaminated areas, respectively. Exposed subjects had significantly elevated levels of As in scalp hair samples (0.72-4.94?μg/g) as compared with referent subjects (0.21-1.484?μg/g; p?相似文献   

Arsenic speciation in human urine reference materials using high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection.

Six arsenic compounds including arsenocholine, arsenobetaine, dimethylarsinic acid, methylarsonic acid, arsenous acid and arsenic acid were separated by high-performance liquid chromatography (HPLC) on a Hamilton PRP-X100 anion-exchange column using isocratic elution and detected by inductively coupled plasma mass spectrometry (ICP-MS). This analytical procedure was applied to the speciation of arsenic compounds in human urine. The influence of urine matrix on the separation of arsenic compounds was evaluated and the determination of arsenic compounds was not hampered by the ArCl interference which has often been encountered in ICP-MS. Three human urine reference materials, SRM 2670 normal level, SRM 2670 elevated level and Lyphocheck urine metal control 1, were analyzed with respect to arsenic compounds by HPLC-ICP-MS. The results were found to be in good agreement with the certified total arsenic concentration in the reference materials. Six arsenic compounds were detected. Arsenobetaine was found to be present in all of the investigated human urine reference materials.     

Methylated arsenic species in plants originate from soil microorganisms

? Inorganic arsenic (iAs) is a ubiquitous human carcinogen, and rice (Oryza sativa) is the main contributor to iAs in the diet. Methylated pentavalent As species are less toxic and are routinely found in plants; however, it is currently unknown whether plants are able to methylate As. ? Rice, tomato (Solanum lycopersicum) and red clover (Trifolium pratense) were exposed to iAs, monomethylarsonic acid (MMA(V)), or dimethylarsinic acid (DMA(V)), under axenic conditions. Rice seedlings were also grown in two soils under nonsterile flooded conditions, and rice plants exposed to arsenite or DMA(V) were grown to maturity in nonsterile hydroponic culture. Arsenic speciation in samples was determined by HPLC-ICP-MS. ? Methylated arsenicals were not found in the three plant species exposed to iAs under axenic conditions. Axenically grown rice was able to take up MMA(V) or DMA(V), and reduce MMA(V) to MMA(III) but not convert it to DMA(V). Methylated As was detected in the shoots of soil-grown rice, and in rice grain from nonsterile hydroponic culture. GeoChip analysis of microbial genes in a Bangladeshi paddy soil showed the presence of the microbial As methyltransferase gene arsM. ? Our results suggest that plants are unable to methylate iAs, and instead take up methylated As produced by microorganisms.     

Effect of arsanilic acid on anaerobic methanogenic process: Kinetics,inhibition and biotransformation analysis

Arsanilic acid (4-aminophenylarsonic acid) is widely used in the poultry and animal industries as a feed additive in the diets. Nearly all the added arsanilic acid is excreted unchanged in manure resulting in the risk of arsenic contamination. In this study, the effects of arsanilic acid on the kinetics, inhibition of methanogenic process and its biotransformation were investigated. The methane yield was not affected by arsanilic acid loading at concentration <0.46 mM, while the methane production was completely inhibited at concentration of 0.92 mM. The IC₅₀ of arsanilic acid in this study was 0.47 mM. After 115 days of incubation, 37–59% of the added arsanilic acid was degraded. The species analysis indicated that at lower initial arsanilic acid concentration, the soluble inorganic arsenic mainly existed in the species of arsenate (As(V)), while at higher initial arsanilic acid concentration (>0.460 mM), the soluble inorganic arsenic mainly existed in the species of arsenite (As(III)), which explains why higher arsanilic acid concentration has severe inhibition to methanogens.     

Distribution of elements binding to molecules with different molecular weights in aqueous extract of Antarctic krill by size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry

The distribution of silver, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium and zinc binding to species with different molecular weight in aqueous extract of krill was studied by on-line size-exclusion chromatography (SEC)/inductively coupled plasma mass spectrometry (ICP-MS). The extract was fractionated in three fractions with different molecular weight (MW) ranges (>20,000 relative molecular mass (rel. mol. mass), 2000-20,000 rel. mol. mass and <2000 rel. mol. mass), which were further analyzed by SEC with columns having different optimum fractionation ranges in order to obtain more detailed information about the MW distribution of the elements. Various distribution profiles for the target elements among different MW ranges were observed. The results obtained indicated that manganese, zinc, silver, cadmium and lead species were mostly distributed in the higher MW range (>20,000 rel. mol. mass). In the case of chromium, iron, cobalt, arsenic and selenium, most of them bind to species with lower MW (<2000 rel. mol. mass). Only copper and nickel species was predominantly present in middle MW range (2000-20,000 rel. mol. mass). Further speciation of arsenic compounds in the small MW fraction was carried out with anion exchange chromatography (AEC) coupled with ICP-MS. The results showed that the dominant arsenic species in this fraction is As(III) (63% of extractable arsenic), while As(V) (13%) and two unknown arsenic species (19% and 5%, respectively) are present in lower amounts.     

Status of Toxic Metals in Biological Samples of Diabetic Mothers and Their Neonates

The mechanism of transport of trace elements from the mother to the newborn is still not well known. The aim of present study was to compare the status of trace toxic elements, arsenic (As), cadmium (Cd), and lead (Pb) in biological samples (whole blood, urine and scalp hair) of insulin-dependent diabetic mothers (age ranged 30-40) and their newly born infants (n = 76). An age and socioeconomics matched 68 nondiabetic mothers and their infants, residing in the same locality, who were selected as referents. The elemental concentrations in all three biological samples were determined by an electrothermal atomic absorption spectrometer, prior to microwave-assisted acid digestion. The mean values of As, Cd, and Pb in all biological samples of diabetic mothers and their infants were significantly higher as compared to the referent mother-infant pair samples (p < 0.01). The high levels of As, Cd, and Pb in biological samples of diabetic women may play a role in the pathogenesis of diabetes mellitus and impacts on their neonates.     

The response of tomato (Lycopersicon esculentum) to different concentrations of inorganic and organic compounds of arsenic

Tomato plants were cultivated in greenhouse and water solutions of arsenite (As(III)), arsenate (As(V)), methylarsonic acid (MA) and dimethylarsinic acid (DMA) were applied individually into cultivation substrate at two As levels, 5 and 15 mg kg⁻¹ of the substrate. Comparing the availability of arsenic compounds increased in order arsenite = arsenate < MA < DMA where the arsenic contents in plants decreased during vegetation period. Within a single plant, the highest arsenic concentration was found in roots followed in decreasing order by leaves, stems, and fruits regardless of arsenic compound applied. Arsenic toxicity symptoms reflected in suppressed growth of plants and a lower number and size of fruits were most significant with DMA treatment. However, the highest accumulation of arsenic by plants growing in the soil containing DMA was caused by higher mobility of this compound in the soil due to its lower sorption affinity. Our results confirmed substantial role of transformation processes of arsenic compounds in soil in uptake and accumulation of arsenic by plants.     

Get phases from arsenic anomalous scattering: de novo SAD phasing of two protein structures crystallized in cacodylate buffer

The crystal structures of two proteins, a putative pyrazinamidase/nicotinamidase from the dental pathogen Streptococcus mutans (SmPncA) and the human caspase-6 (Casp6), were solved by de novo arsenic single-wavelength anomalous diffraction (As-SAD) phasing method. Arsenic (As), an uncommonly used element in SAD phasing, was covalently introduced into proteins by cacodylic acid, the buffering agent in the crystallization reservoirs. In SmPncA, the only cysteine was bound to dimethylarsinoyl, which is a pentavalent arsenic group (As (V)). This arsenic atom and a protein-bound zinc atom both generated anomalous signals. The predominant contribution, however, was from the As anomalous signals, which were sufficient to phase the SmPncA structure alone. In Casp6, four cysteines were found to bind cacodyl, a trivalent arsenic group (As (III)), in the presence of the reducing agent, dithiothreitol (DTT), and arsenic atoms were the only anomalous scatterers for SAD phasing. Analyses and discussion of these two As-SAD phasing examples and comparison of As with other traditional heavy atoms that generate anomalous signals, together with a few arsenic-based de novo phasing cases reported previously strongly suggest that As is an ideal anomalous scatterer for SAD phasing in protein crystallography.