Combination of HPLC with organic and inorganic mass spectrometry to study the metabolic response of the clam Scrobicularia plana to arsenic exposure

Arsenic is a toxic element extensively studied in the marine environment due to differential toxicological effects of inorganic and organic species. In the present work, the bivalve Scrobicularia plana was exposed to As^V (10 and 100 μg/L) for 14 days to evaluate the metabolic perturbations caused by this element. Arsenic speciation and metabolomic analysis were performed in the digestive gland of the bivalve using two complementary analytical platforms based on inorganic and organic mass spectrometry. It has been observed the greater presence of the innocuous specie arsenobetaine produced in this organism as defense mechanism against arsenic toxicity, although significant concentrations of methylated and inorganic arsenic were also present, depending on the level of arsenic in aqueous media. Complementarily, a metabolomic study based on mass spectrometry and statistical discriminant analysis allows a good classification of samples associated to low and high As(V) exposure in relation to controls. About 15 metabolites suffer significant changes of expression by the presence of As(V): amino acids, nucleotides, energy‐related metabolites, free fatty acids, phospholipids and triacylglycerides, which can be related to membrane structural and functional damage. In addition, perturbation of the methylation cycle, associated with the increase of homocysteine and methionine was observed, which enhance the methylation of toxic inorganic arsenic to less toxic dimethylarsenic.     

Urinary arsenic speciation by high-performance liquid chromatography/atomic absorption spectrometry for monitoring occupational exposure to inorganic arsenic

Total urinary arsenic determinations are often used to assess occupational exposure to inorganic arsenic. Ingestion of sea food can increase the normal background levels of total arsenic in urine by up to an order of magnitude, but this arsenic has relatively little toxicity; it is tightly bound as arsenobetaine. The excretion of inorganic arsenic and its metabolites dimethylarsenic acid (DMA) and monomethylarsonic acid (MMA) is not influenced by the consumption of arsenic from sea food. Specific measurements of DMA, MMA and inorganic arsenic provide a more reliable indicator or exposure than total urinary arsenic levels. An automated atomic absorption method involving high-performance liquid chromatographic separation of the arsenic species and continuous hydride generation is described for the determination of arsenite, arsenate, DMA and MMA at μg As l^?1 levels. The method is used to study normal urinary arsenic levels in laboratory staff and arsenic excretion by exposed workers.     

Methylated arsenic species in estuarine porewaters

Inorganic arsenic, monomethylarsenic and dimethylarsenic species have been observed in samples of sediment porewater collected from the Tamar Estuary in South-West England. Porewater samples were collected using in situ dialysis. The arsenic species were separated by hydride generation and concentrated by liquid nitrogen trapping, prior to analysis by directly coupled gas chromatography-atomic absorption spectroscopy. The predominant dissolved arsenic species present was inorganic arsenic (5-62 m?g dm^?3). However, this is the first time significant concentrations of methylated arsenic species have been quantified in estuarine porewaters (0.04–0.70 m?g dm^?3), accounting for between 1 and 4% of the total dissolved arsenic. The presence of methylated arsenic compounds in porewaters is attributed to in situ environmental methylation, although the possibility of methylated arsenic species being derived from biological debris cannot be excluded.     

Species differences in the metabolism of arsenic compounds

Humans are exposed via air, water and food to a number of different arsenic compounds, the physical, chemical, and toxicological properties of which may vary considerably. In people eating much fish and shellfish the intake of organic arsenic compounds, mainly arsenobetaine, may exceed 1000 μg As per day, while the average daily intake of inorganic arsenic is in the order of 10–20 μg in most countries. Arsenobetaine, and most other arsenic compounds in food of marine origin, e.g. arsenocholine, trimethylarsine oxide and methylarsenic acids, are rapidly excreted in the urine and there seem to be only minor differences in metabolism between animal species. Trivalent inorganic arsenic (AsIII) is the main form of arsenic interacting with tissue constituents, due to its strong affinity for sulfhydryl groups. However, a substantial part of the absorbed AsIII is methylated in the body to less reactive metabolities, methylarsonic acid (MMA) and dimethylarsinic acid (DMA), which are rapidly excreted in the urine. All the different steps in the arsenic biotransformation in mammals have not yet been elucidated, but it seems likely that the methylation takes place mainly in the liver by transfer of methyl groups from S-adenosylmethionine to arsenic in its trivalent oxidation state. A substantial part of absorbed arsenate (AsV) is reduced to AsIII before being methylated in the liver. There are marked species differences in the methylation of inorganic arsenic. In most animal species DMA is the main metabolite. Compared with human subjects, very little MMA is produced. The marmoset monkey is the only species which has been shown unable to methylate inorganic arsenic. In contrast to other species, the rat shows a marked binding of DMA to the hemoglobin, which results in a low rate of urinary excretion of arsenic.     

Determination of inorganic and methylated arsenic species in marine organisms and sediments

The determination of inorganic arsenic, monomethylarsenic and dimethylarsenic in marine organisms and estuarine sediments is described. The arsenic species are isolated by solvent extraction, separated by ion-exchange chromatography and selectively determined by aisine generation. Recoveries of spikes of 5 and 10 μg of arsenic taken through the whole procedure were 92–96%. Typical results obtained in a study of the forms of arsenic in several species of macroalgae, tissues of Mercenaria mercenaria and estuarine sediments are given.     

Biomethylation of Arsenic in an Arsenic-rich Freshwater Environment

Arsenic circulation in an arsenic-rich freshwater ecosystem was elucidated to detect arsenic species in the river water and in biological samples living in the freshwater environment. Water-soluble arsenic compounds in biological samples were extracted with 70% methanol. Samples containing arsenic compounds in the extracts were treated with 2 mol dm³ of sodium hydroxide and reduced with sodium borohydride. The detection of arsenic species was accomplished using a hydride generation/cold trap/cryofocus/gas chromatography-mass spectrometry (HG/CT/CF/GC-MS) system. The major arsenic species in the river water, freshwater algae and fish are inorganic arsenic, dimethylarsenic and trimethylarsenic compounds, respectively. Trimethylarsenic compounds are also detected in aquatic macro-invertebrates. The freshwater unicellular alga Chlorella vulgaris, in a growth medium containing arsenate, accumulated arsenic and converted it to a dimethylarsenic compound. The water flea Daphnia magna, which was fed on arsenic-containing algae, converted it to a trimethylarsenic species. © 1997 by John Wiley & Sons, Ltd.     

Seasonal control of arsenic speciation in an estuarine ecosystem

Arsenic speciation in the Itchen estuary and Southampton Water (UK) has been shown to vary seasonally, with detectable (>0.02μg As dm^?3) dissolved arsenic(III) and methylated arsenic only being present from May to early October. This corresponds to the time period during which water temperatures exceed 12°C. For the remainder of the year, inorganic arsenic(V) was the only detectable species. At its peak, ca 30% of the dissolved arsenic was present as methylated forms with dimethylarsenic (DMAs) being the predominant bioarsenical. Significant quantities of monomethyl-arsenic (MMAs) and inorganic arsenic(III) were also present, however. The concentrations of the bioarsenical species varied with position in the estuary and generally increased with salinity. Measurements made during the period of peak algal activity implicated the highsalinity area of the estuary as the most probable region in which the methylated arsenicals are generated. At some sites, a distinct lag was observed between the appearance of dimethylarsenic and the detection of arsenic(III)and monomethylarsenic. Chlorophyll a concentration proved to be a poor predictor of the appearance of reduced and methylated arsenic in the water column. Possible sources of dissolved methylated arsenic are discussed.     

Hydride-trapping techniques for the speciation of arsenic

The determination of arsenic species by the trapping of volatile hydrides prior to atomization in the light path of an atomic absorption spectrometer is described and its operation in the measurement of arsenic species in the marine environment are discussed. Examples are drawn from the analysis of Tamar estuary water and sediment interstitial (pore) waters and from studies of the temporal variation of dimethylarsenic in coastal waters. Improvements in both the design and operation of the technique have resulted in enhanced performance. Baseline resolution of inorganic arsenic, monomethylarsenic and dimethylarsenic is now possible and trimethylarsine is resolved. Ultraviolet photolysis of arsenobetaine and arsenocholine gives partial conversion to trimethylarsine oxide. This can be employed in the qualitative appraisal of the presence of trimethylarsenic species. Current detection limits (3 sigma) for inorganic, mono- and di-methylarsenic lie in the range 19 to 61 pg absolute, giving 19–61 ng/1 concentration detection limits for 1 ml samples. This can be improved even further by using larger sample volumes. The properties of the analysis system when presented with various arsenic species are described. A ca. 10% loss of arsenite occurs in samples stored at —20 °C and immediate freezing of samples in liquid nitrogen is recommended.     

Metabolomics of arsenic based on speciation studies

Biomedical research on arsenic can be divided into three steps, i.e., speciation of the entirety of arsenic in a biological system (metallome), examination of the metabolism of arsenic based on the speciation of metallome (metabolomics), and examination of the metallomics underlying the mechanism that triggers biological/physiological/toxicological effects based on the metabolomics. In the present communication, the metabolic pathway for inorganic arsenic, a known human carcinogen, was explained based on current results of speciation. In addition to the consecutive reduction and oxidative methylation reactions converting inorganic arsenicals to the major urinary metabolite dimethylarsinic acid, the role of the conjugation reaction involving glutathione resulting in excretion from the liver was discussed. Furthermore, sulfur-containing arsenicals (thioarsenicals) identified as new metabolites in the livers of rats were characterized chemically and metabolically.     

Generation of AsH(3) from As(V) in the absence of KI as prereducing agent: Speciation of inorganic arsenic

It is shown that the potassium iodide to the samples to reduce As(V) to AS(III) is not essential when total inorganic arsenic is determined by molecular spectrophotometry (trapping AsH(3) in Ag-DDTC) or by atomic-absorption spectrometry (if Ar flow-rate and NaBH(4) addition rate are controlled in 6M hydrochloric acid medium). Furthermore, in the presence of low concentration of organic arsenic, a method is reported for the selective determination of inorganic As(III) and As(V), based on the use of citrate/citric acid medium to determine As(III) and hydrochloric acid to determine total inorganic As. As(V) is determined by the difference between total inorganic As and As(III). The interference level of organic arsenic species (monomethylarsenic acid and dimethylarsenic acid) in the determination of total inorganic arsenic and AS(III) in 6M hydrochloric acid and citrate/citric acid medium respectively, is reported in the text. The developed method is applied to determine As(III) and As(V) in spiked, tap and waste waters and in lake sediments.     

Quantitative measurements of inorganic and organic arsenic by flameless atomic absorption spectrometry

Procedures are described for the determination of arsenicals in water and urine by flameless atomic absorption spectrometry ; these avoid the isolation and transfer of arsine(s) and permit some differentiation between the inorganic and organic (methyl) arsenic content of a sample. Samples of water or urine are heated with hydrochloric acid, and treated with iodide ion. Arsenic species, as the iodides, are extracted into chloroform and then either reextracted into deionized water for measurement of inorganic arsenic, or reextracted into dilute dichromate solution for total arsenic determination; the difference furnishes levels of organic arsenic. Aliquots of the final aqueous extracts are analyzed by graphite-furnace atomic absorption spectrometry, with an arsenic electrodeless discharge lamp. The lower detection limit for water and urine was 10 p.p.b. The recoveries (and Sg values) were: 87.0% (3.0) and 93.0 % (7.9), for inorganic arsenic in water and urine, respectively; 92.3 % (5.3) for mixtures of inorganic and methylated arsenic (total arsenic) in water and urine; and 98.7 % (3.9) and 88.4% (3.6) for dimethylarsenic in water and urine, respectively.     

Identification of arsenic species in sheep‐wool extracts by different chromatographic methods

Sheep on the island of North Ronaldsay (Orkney, UK) feed mostly on seaweed, which contains high concentrations of dimethylated arsenoribosides. Wool of these sheep contains dimethylated, monomethylated and inorganic arsenic, in addition to unidentified arsenic species in unbound and complexed form. Chromatographic techniques using different separation mechanisms and detectors enabled us to identify five arsenic species in water extracts of wool. The wool contained 5.2 ± 2.3 µg arsenic per gram wool. About 80% of the arsenic in wool was extracted by boiling the wool with water. The main species is dimethylarsenic, which accounted for about 75 to 85%, monomethylated arsenic at about 5% and the rest is inorganic arsenic. Depending on the separation method and condition, the chromatographic recovery of arsenic species was between 45% for the anion exchange column, 68% for the size exclusion chromatography (SEC) and 82% for the cation exchange column. The SEC revealed the occurrence of two unknown arsenic compounds, of which one was probably a high molecular mass species. Since chromatographic recovery can be improved by either treating the extract with CuCl/HCl (CAT: 90%) or longer storage of the sample (CAT: 105%), in particular for methylated arsenic species, it can be assumed that labile arsenic–protein‐like coordination species occur in the extract, which cannot be speciated with conventional chromatographic methods. It is clear from our study of sheep wool that there can be different kinds of ‘hidden’ arsenic in biological matrices, depending on the extraction, separation and detection methods used. Hidden species can be defined as species that are not recordable by the detection system, not extractable or do not elute from chromatographic columns. Copyright © 2003 John Wiley & Sons, Ltd.     

Arsenic metabolism in seaweed-eating sheep from Northern Scotland

Cation exchange and anion exchange liquid chromatography were coupled to an ICP-MS and optimised for the separation of 13 different arsenic species in body fluids (arsenite, arsenate, dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), trimethylarsine oxide (TMAO), tetramethylarsonium ion (TMA), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinoyl ethanol (DMAE) and four common dimethylarsinoylribosides (arsenosugars). The arsenic species were determined in seaweed extracts and in the urine and blood serum of seaweed-eating sheep from Northern Scotland. The sheep eat 2-4 kg of seaweed daily which is washed ashore on the most northern Island of Orkney. The urine, blood and wool of 20 North Ronaldsay sheep and kidney, liver and muscle from 11 sheep were sampled and analysed for their arsenic species. In addition five Dorset Finn sheep, which lived entirely on grass, were used as a control group. The sheep have a body burden of approximately 45-90 mg arsenic daily. Since the metabolism of arsenic species varies with the arsenite and arsenate being the most toxic, and organoarsenic compounds such as arsenobetaine the least toxic compounds, the determination of the arsenic species in the diet and their body fluids are important. The major arsenic species in their diet are arsenoribosides. The major metabolite excreted into urine and blood is DMAA (95 +/- 4.1%) with minor amounts of MMAA, riboside X, TMA and an unidentified species. The occurrence of MMAA is assumed to be a precursor of the exposure to inorganic arsenic, since demethylation of dimethylated or trimethylated organoarsenic compounds is not known (max. MMAA concentration 259 microg/L). The concentrations in the urine (3179 +/- 2667 microg/L) and blood (44 +/- 19 microg/kg) are at least two orders of magnitude higher than the level of arsenic in the urine of the control sheep or literature levels of blood for the unexposed sheep. The tissue samples (liver: 292 +/- 99 microg/kg, kidney: 565 +/- 193 microg/kg, muscle: 680 +/- 224 microg/kg) and wool samples (10470 +/- 5690 microg/kg) show elevated levels which are also 100 times higher than the levels for the unexposed sheep.     

Speciation of major arsenic species in seawater by flow injection hydride generation atomic absorption spectrometry

Arsenic present at 1 microg L(-1) concentrations in seawater can exist as the following species: As(III), As(V), monomethylarsenic, dimethylarsenic and unknown organic compounds. The potential of the continuous flow injection hydride generation technique coupled to atomic absorption spectrometry (AAS) was investigated for the speciation of these major arsenic species in seawater. Two different techniques were used. After hydride generation and collection in a graphite tube coated with iridium, arsenic was determined by AAS. By selecting different experimental hydride generation conditions, it was possible to determine As(III), total arsenic, hydride reactive arsenic and by difference non-hydride reactive arsenic. On the other hand, by cryogenically trapping hydride reactive species on a chromatographic phase, followed by their sequential release and AAS in a heated quartz cell, inorganic As, MMA and DMA could be determined. By combining these two techniques, an experimental protocol for the speciation of As(III), As(V), MMA, DMA and nonhydride reactive arsenic species in seawater was proposed. The method was applied to seawater sampled at a Mediterranean site and at an Atlantic coastal site. Evidence for the biotransformation of arsenic in seawater was clearly shown.     

Speciation of major arsenic species in seawater by flow injection hydride generation atomic absorption spectrometry

Arsenic present at 1 μg L^–1 concentrations in seawater can exist as the following species: As(III), As(V), monomethylarsenic, dimethylarsenic and unknown organic compounds. The potential of the continuous flow injection hydride generation technique coupled to atomic absorption spectrometry (AAS) was investigated for the speciation of these major arsenic species in seawater. Two different techniques were used. After hydride generation and collection in a graphite tube coated with iridium, arsenic was determined by AAS. By selecting different experimental hydride generation conditions, it was possible to determine As(III), total arsenic, hydride reactive arsenic and by difference non-hydride reactive arsenic. On the other hand, by cryogenically trapping hydride reactive species on a chromatographic phase, followed by their sequential release and AAS in a heated quartz cell, inorganic As, MMA and DMA could be determined. By combining these two techniques, an experimental protocol for the speciation of As(III), As(V), MMA, DMA and non-hydride reactive arsenic species in seawater was proposed. The method was applied to seawater sampled at a Mediterranean site and at an Atlantic coastal site. Evidence for the biotransformation of arsenic in seawater was clearly shown.     

Arsenic metabolism in seaweed-eating sheep from Northern Scotland

Cation exchange and anion exchange liquid chromatography were coupled to an ICP-MS and optimised for the separation of 13 different arsenic species in body fluids (arsenite, arsenate, dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), trimethylarsine oxide (TMAO), tetramethylarsonium ion (TMA), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinoyl ethanol (DMAE) and four common dimethylarsinoylribosides (arsenosugars). The arsenic species were determined in seaweed extracts and in the urine and blood serum of seaweed-eating sheep from Northern Scotland. The sheep eat 2–4 kg of seaweed daily which is washed ashore on the most northern Island of Orkney. The urine, blood and wool of 20 North Ronaldsay sheep and kidney, liver and muscle from 11 sheep were sampled and analysed for their arsenic species. In addition five Dorset Finn sheep, which lived entirely on grass, were used as a control group. The sheep have a body burden of approximately 45–90 mg arsenic daily. Since the metabolism of arsenic species varies with the arsenite and arsenate being the most toxic, and organoarsenic compounds such as arsenobetaine the least toxic compounds, the determination of the arsenic species in the diet and their body fluids are important. The major arsenic species in their diet are arsenoribosides. The major metabolite excreted into urine and blood is DMAA (95 ± 4.1%) with minor amounts of MMAA, riboside X, TMA and an unidentified species. The occurrence of MMAA is assumed to be a precursor of the exposure to inorganic arsenic, since demethylation of dimethylated or trimethylated organoarsenic compounds is not known (max. MMAA concentration 259 μg/L). The concentrations in the urine (3179 ± 2667 μg/L) and blood (44 ± 19 μg/kg) are at least two orders of magnitude higher than the level of arsenic in the urine of the control sheep or literature levels of blood for the unexposed sheep. The tissue samples (liver: 292 ± 99 μg/kg, kidney: 565 ± 193 μg/kg, muscle: 680 ± 224 μg/kg) and wool samples (10 470 ± 5690 μg/kg) show elevated levels which are also 100 times higher than the levels for the unexposed sheep. Received: 29 February 2000 / Revised: 26 April 2000 / Accepted: 1 May 2000     

Interpretation of inorganic arsenic metabolism in humans in the light of observations made in vitro and in vivo in the rat

The toxicity of inorganic trivalent arsenic for living organisms is reduced by in vivo methylation of the element. In man, this biotransformation leads to the synthesis of monomethylarsonic (MMA) and dimethylarsinic (DMA) acids, which are efficiently eliminated in urine along with the unchanged form (As_i). In order to document the methylation process in humans, the kinetics of As_i, MMA and DMA elimination were studied in volunteers given a single dose of one of these three arsenicals or repeated doses of As_i. The arsenic methylation efficiency was also assessed in subjects acutely intoxicated with arsenic trioxide (As₂O₃) and in patients with liver diseases. Several observations in humans can be explained by the properties of the enzymic systems involved in the methylation process which we have characterized in vitro and in vivo in rats as follows: (1) production of As_i metabolites is catalyzed by an enzymic system whose activity is highest in liver cytosol; (2) different enzymic activities, using the same methyl group donor (S-adenosylmethionine), lead to the production of mono- and di-methylated derivatives which are excreted in urine as MMA and DMA; (3) dimethylating activity is highly sensitive to inhibition by excess of inorganic arsenic; (4) reduced glutathione concentration in liver moderates the arsenic methylation process through several mechanisms, e.g. stimulation of the first methylation reaction leading to MMA, facilitation of As_i uptake by hepatocytes, stimulation of the biliary excretion of the element, reduction of pentavalent forms before methylation, and protection of a reducing environment in the cells necessary to maintain the activity of the enzymic systems.     

Intake of different chemical species of dietary arsenic by the japanese,and their blood and urinary arsenic levels

We calculated the intake of each chemical species of dietary arsenic by typical Japanese, and determined urinary and blood levels of each chemical species of arsenic. The mean total arsenic intake by 35 volunteers was 195±235 (15.8-1039) μg As day^?1, composed of 76% trimethylated arsenic (TMA), 17.3% inorganic arsenic (As_i), 5.8% dimethylated arsenic (DMA), and 0.8% monomethylated arsenic (MA): the intake of TMA was the largest of all the measured species. Intake of As_i characteristically and invariably occurred in each meal. Of the intake of As_i, 45-75% was methylated in vivo to form MA and DMA, and excreted in these forms into urine. The mean measured urinary total arsenic level in 56 healthy volunteers was 129±92.0 μg As dm^?3, composed of 64.6% TMA, 26.7% DMA, 6.7% As_i and 2.2% MA. The mean blood total arsenic level in the 56 volunteers was 0.73±0.57 μg dl^?1, composed of 73% TMA, 14% DMA and 9.6% As_i. The urinary TMA levels proved to be significantly correlated with the whole-blood TMA levels (r = 0.376; P<0.01).     

Simulate methylation reaction of arsenic(III) with methyl iodide in an aquatic system

The methylation reaction of inorganic arsenic occurring in aquatic systems was studied by HPLC‐HG‐AFS as a method to separate and detect soluble methylarsenic species. Transformation from inorganic arsenic to methylarsenic was essential for major changes in toxicity to organisms. Monomethylarsenic [AsOCH₃(OH)₂] was the only product in the methylation reaction of inorganic arsenic(III) with methyl iodide (MeI). This process can be described as an oxidative carbonium‐ion transfer, with MeI acting as a methyl donor. From a thermodynamic point of view, the activity of the carbonium ion and pH were the two major influencing factors. The pH dependence of redox potential of As(III) was the reason for the effect of pH on methylation of arsenic. The influences of salinity and concentration of the methyl donor may be explained by their effects on the activity of carbonium. Moreover, kinetics experiments demonstrated that the methylation reaction was first‐order for both As(III) and methyl iodide. First‐order reaction rates were also calculated at different pH, salinity and MeI, and were found to be in the range 0.0026–0.0123 h^?1. The methylation rate varied largely under different reaction conditions. Copyright © 2005 John Wiley & Sons, Ltd.     

Conversion of arsenite and arsenate to methylarsenic and dimethylarsenic compounds by homogenates prepared from livers and kidneys of rats and mice

Pooled livers and pooled kidneys from rats or mice were homogenized and spiked with arsenite or arsenate in the concentration range 1.3–20 μmol dm^?3. Methylarsenic and dimethylarsenic compounds were determined by the hydride generation technique in the homogenates after a 90 min incubation at 37°C. The rat homogenates methylated arsenite and arsenate more efficiently than the mouse homogenates. Monomethylated arsenic was present in larger amounts than dimethylated arsenic in the rat homogenates. In the absence of reduced glutathione (GSH), no methylation occurred. Addition of GSH promoted monomethylation and dimethylation, whereas dithiothreitol and mercaptoethanol (10 mmol dm^?3) fostered only monomethylation. The amounts of monomethylated arsenic in the rat liver homogenates increased with increasing arsenite concentration (1.3–20 μmol dm^?3) however, the percentage of arsenic that had been methylated decreased. A similar trend, but with much less monomethylarsenic formed, was observed for arsenate-spiked homogenates. Rat kidney homogenates methylated arsenite and arsenate to a much smaller extent than rat liver homogenates. The K_m values for the monomethylation in rat liver homogenates were found to be 5.3 μmol dm^?3 for arsenite and 59 μmol dm^?3 for arsenate.