Determination of dopamine in the presence of ascorbic acid using poly(3,5-dihydroxy benzoic acid) film modified electrode

A polymerized film of 3,5-dihydroxy benzoic acid (DBA) was prepared on the surface of a glassy carbon electrode (GCE) in neutral solution by cyclic voltammetry (CV). The poly(DBA) film-coated GCE exhibited excellent electrocatalytic activity toward the oxidation of dopamine (DA). A linear range of 1.0 × 10⁻⁷ to 1.0 × 10⁻⁴ M and a detection limit of 6.0 × 10⁻⁸ M were observed in pH 7.4 phosphate buffer solutions. Moreover, the interference of ascorbic acid (AA) was effectively eliminated. This work provides a simple and easy approach to selective detection of DA in the presence of AA.     

Correlation between the presence of Gonyaulax fragilis (Dinophyceae) and the mucilage phenomena of the Emilia-Romagna coast (northern Adriatic Sea)

The formation of massive amounts of suspended mucilaginous organic matter which periodically affects the Adriatic Sea, has been regarded as a complex physico-chemical phenomenon resulting from the production of extracellular material by phytoplankton. Although the exact cause has remained obscure, the mechanism of its formation has usually been considered to be a long-term process, starting after the late winter-early spring blooms, and involving the participation of various algal species, mainly within the diatom group. In this paper we report on the results of a phytoplankton monitoring programme in northern Adriatic seawaters off the Emilia-Romagna coast of Italy which revealed the constant concomitant presence of the dinoflagellate Gonyaulax fragilis (Schütt) Kofoid and mucilaginous formations. In the early stages of the phenomenon the dinoflagellate was clearly observable by microscopic examination in the mucilage, but as the mucilage aged this alga almost completely decomposed and diatom cells increased in number and became predominant. Although characterized by a slow growth rate in culture, in natural seawater G. fragilis was observed to reach cell densities of up to 7.0×10⁶ cells l⁻¹. The results of this study lead us to propose the hypothesis that the appearance of mucilage in the water column of the Adriatic Sea is the consequence of a seasonal growth of this dinoflagellate favoured by specific environmental circumstances.     

Genotoxicity is modulated by ascorbic acid: Studies using the wing spot test in Drosophila

The ability of ascorbic acid (Vitamin C) to modulate the genotoxic action of several mutagens was investigated in the wing spot test of Drosophila melanogaster. In this assay, 3-day-old transheterozygous larvae for the multiple wing hairs (mwh, 3-0.3) and flare (flr, 3-38.8) genes were treated with three reference mutagenic compounds, namely cobalt chloride (CoCl₂), 4-nitroquinoline 1-oxide (4-NQO) and potassium dichromate (K₂Cr₂O₇). The results obtained show that the three reference mutagens tested were clearly genotoxic in the Drosophila wing somatic mutation and recombination test (SMART). None of the three concentrations tested of ascorbic acid (25, 75 and 250 mM) induced significant increases in the frequency of the mutant clones recorded. When co-treatment experiments with ascorbic acid were carried out, different results were found. Thus, ascorbic acid was effective in reducing the genotoxicity of K₂Cr₂O₇ virtually to the control level; on the contrary, it did not show any antigenotoxic effect on the genotoxicity of 4-NQO. Finally, co-treatments with CoCl₂ and ascorbic acid show a significant increase in the frequency of mutant clones over the values obtained with CoCl₂ alone.     

Synthesis of super-high-molecular-weight poly-γ-glutamic acid by Bacillus subtilis subsp. chungkookjang

Poly-γ-glutamic acid (PGA) with high molecular weight is a most promising biomaterial in industrial uses; however, it generally diverse in molecular structure and co-produced with polysaccharides and various other biopolymers. In this study, it was ascertained that Bacillus subtilis subsp. chungkookjang cells are superior to B. subtilis (natto) cells as the biocatalyst for the synthesis of super-high-molecular-weight PGA (over 2000 k). We effectively purified PGA and fractionated according to its molecular weight by anion-exchange chromatography, and further developed a simple method for determination of the molecular weight of PGA on the basis of numbers of glutamate monomers generated by hydrolysis and a free amino group quantified with 1-fluoro-2,4-dinitrobenzene (FDNB). The molecular weight determination with FDNB was available even for a super-high-molecular-weight PGA, e.g. the 2000-k polymer. Super-high-molecular-weight PGAs (average 2000 k and 7000 k), which were synthesized by the use of B. subtilis subsp. chungkookjang cells in the presence of a high concentration of ammonium sulfate, were rich in l-glutamate rather than in the d-enantiomer.     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

Selective detection of uric acid in the presence of ascorbic acid at physiological pH by using a beta-cyclodextrin modified copolymer of sulfanilic acid and N-acetylaniline

A beta-cyclodextrin (CD) modified copolymer membrane of sulfanilic acid (p-ASA) and N-acetylaniline (SPNAANI) on glassy carbon electrode (GCE) was prepared and used to determine uric acid (UA) in the presence of a large excess of ascorbic acid (AA) by differential pulse voltammetry (DPV). The properties of the copolymer were characterized by X-ray photoelectron spectra (XPS) and Raman spectroscopy. The oxidation peaks of AA and UA were well separated at the composite membrane modified electrode in phosphate buffer solution (PBS, pH 7.4). A linear relationship between the peak current and the concentration of UA was obtained in the range from 1.0 x 10(-5) to 3.5 x 10(-4)mol L(-1), and the detection limit was 2.7 x 10(-6)mol L(-1) at a signal-to-noise ratio of 3. Two hundred and fifty-fold excess of AA did not interfere with the determination of UA. The application of the prepared electrode was demonstrated by measuring UA in human serum samples without any pretreatment, and the results were comparatively in agreement with the spectrometric clinical assay method.     

CV and NMR study on the reaction of Mo(VI) with 3,4-dihydroxybenzoic acid and ascorbic acid in aqueous solution

The gradual release of the ligand 3,4-dihydroxybenzoic acid (3,4-DHBA) from its molybdenum complex in the presence of ascorbic acid (AscA) in a weakly acidic aqueous solution (pH ∼ 3.5) is described. We observed that the formation of the 3,4-DHBA-semiquinone oxidation state and the semidehydroascorbate is a pre-requisite for the release of the 3,4-DHBA ligand. The interaction of these radicals leads at the same time to the further degradation of AscA resulting in, among other compounds, threonic acid which participates in the reaction with molybdenum. The comparison of the complexing ability indicated that threonic acid competes with protocatechuate, while ascorbic acid is a less good ligand for the Mo(VI). Solution studies on the reaction mechanism were performed by cyclic voltammetry, NMR spectroscopy and UV-Vis spectroscopy. Isolated precipitates were investigated by NMR spectroscopy. The antioxidant properties of 3,4-DHBA and AscA were also compared using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH).     

Highly sensitive and selective method to detect dopamine in the presence of ascorbic acid by a new polymeric composite film

A new approach was applied to modify gold electrode with a unique polymer composite for selectively detecting dopamine (DA), a neurotransmitter, in the presence of an electroactive species of ascorbic acid (AA). After self-assembly of 11-mercaptoundecanoic acid (MUA) monolayer on gold surface, polyethylene glycol (PEG) was used to perform electrochemical esterification with MUA. In general, AA is the main interference of DA detection in a biological system. The resulting composite layer showed high sensitivity to detect DA but selectively blocked the interference from AA. Furthermore, for the first time, an interesting mechanism was demonstrated from our experimental results, namely, that the catalytic effect of AA on DA is limited by DA concentration when AA/DA>1. The modified electrode showed good reproducibility (+/-2% relative standard deviation), a low detection limit (10 nM), a fast response time (<2s), high sensitivity (86 nA/microM), a wide dynamic range of detection (20 microM), and great selectivity (without AA interference). The discovery is very promising for applications of detection of DA in a physiological environment where a high concentration of AA always exists.     

Preparation of dehydro-L-ascorbic acid dimer by air oxidation of L-ascorbic acid in the presence of catalytic amounts of copper(II) acetate and pyridine

The catalytic system Cu(AcO)2-pyridine 1:4 mol% in methanol, slowly catalyses the air oxidation of ascorbic acid to the 2-methyl hemi-ketal of dehydroascorbic acid 5, and hydrogen peroxide. However, with Cu(AcO)2-pyridine 3:4 mol% the air oxidation is quite fast and no hydrogen peroxide is present at the end of the reaction. Removal of the catalyst and refluxing the foamy 5 in MeCN gives the oxidized, dimeric, dehydroascorbic acid in very good yields (approximately 70%) contaminated by approximately 1-2% MeCN.     

Differential distribution of ascorbic acid,peroxidase activity,and hydrogen peroxide along the root axis in Allium cepa L. and its possible relationship with cell growth and differentiation

Summary.  In this paper we show an asymmetrical distribution of apoplastic and symplastic ascorbic acid content, peroxidase activities and hydrogen peroxide along the root axis in Allium cepa L. For most of these metabolites, a marked gradient from the root apex to the onion base was observed and was different for apoplastic and symplastic compartments. In total homogenates, ascorbic acid content was higher in the zones closer to the apex and decreased towards the root base. However, an opposite pattern was observed in the apoplastic fraction. Peroxidase activities with guaiacol, ferulic acid, ascorbic acid, and coniferyl alcohol were also different depending on the evaluated zone and the fraction used (apoplastic or symplastic). In general, each activity had a specific and unique pattern. Immunodetection of peroxidase proteins in Western blots using anti-horseradish peroxidase and anti-ascorbate peroxidase antibodies revealed different bands at the different zones of the root. Hydrogen peroxide was detected by electron microscopy and was mainly found in cell walls of epidermis (or rhizodermis), meristem, and elongating cells. The number of cell walls showing hydrogen peroxide decreased dramatically towards the root base. The results suggest that the different zones of the root show specific requirements for ascorbic acid and hydrogen peroxide. Also, each fragment of the root seems to express specific peroxidase proteins. Different processes that take place at every part of the root, as cell proliferation and elongation near the root apex and gradual lignification and differentiation towards the root base are the key to explain the results. Received May 10, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Edificio C-6, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Domoic acid production by Pseudo-nitzschia calliantha Lundholm, Moestrup et Hasle (bacillariophyta) isolated from the Black Sea

A species of Pseudo-nitzschia isolated from Sevastopol Bay, Black Sea, was examined for its toxicity. The species was identified as P. calliantha Lundholm, Moestrup et Hasle based on SEM and TEM examination. Domoic acid (DA) was detected in batch culture throughout the growth cycle of P. calliantha. The production of DA by this diatom species was confirmed by fluorenylmethoxycarbonyl (FMOC) derivatization and HPLC-fluorescence method. The cellular DA level was higher in the early exponential phase, with the maximum value of 0.95 pg DA cell⁻¹. In the stationary phase, the cellular DA levels declined. This is the first record of a DA producing diatom isolated from the Black Sea.     

Purification, characterisation, and inhibition by monoterpenes of acetylcholinesterase from the waxmoth, Galleria mellonella (L.)

Acetylcholinesterase (AChE) was purified from the brain of the waxmoth, Galleria mellonella (L.) by affinity chromatography followed by anion exchange chromatography. It resolved as a single band by polyacrylamide gel electrophoresis (PAGE) both non-denaturing and SDS (silver stained), and as a single peak by high pressure liquid chromatography (HPLC), in an overall yield of 32% representing 283-fold purification. This was a true acetylcholinesterase, with no activity as a non-specific cholinesterase (butyrylcholinesterase). The molecular weight determined by PAGE in the absence and presence of sodium dodecyl sulphate (SDS) was ca. 240,000 Da and 60,000 Da respectively, indicating an arrangement of tetrameric subunits. 2-Heptanone, the honeybee alarm pheromone, reversibly and competitively inhibited the purified Galleria AChE with a K_i value of 1.34×10⁻³ M. Furthermore, five monoterpenes associated with plant defence and representing a range of functional groups, also were reversible competitive inhibitors of the purified AChE from Galleria, which is consistent with previous data for electric eel AChE (Ryan, M.F., Byrne, O., 1988. Plant-insect coevolution and inhibition of acetylcholinesterase. Journal of Chemical Ecology 14, 1965-1975).     

Redox Properties of Iron in the Binding Site(s) of F1ATPase from Mammalian Mitochondria and Thermophilic Bacterium PS3: A Comparative Study

Iron ions in the two iron centers of beef heart mito-chondrial F, ATPase, which we have been recently characterized (FEBS Letters 1996,379, 231-235), exhibit different redox properties. In fact, the ATP-dependent site is able to maintain iron in the redox state of Fe(II) even in the absence of reducing agents, whereas in the nucleotide-independent site iron is oxidized to Fe(III) upon removal of the reductant. Fe(III) ions in the two sites display different reactivity towards H₂O₂, because only Fe(III) bound in the nucleotide-independent site rapidly reacts with H₂O₂ thus mediating a 30% enzyme inactivation. Thermophilic bacterium PS3 bears one Fe(III) binding site, which takes up Fe(III) either in the absence or presence of nucleotides and is unable to maintain iron in the redox state of Fe(II) in the absence of ascorbate. Fe(III) bound in thermophilic F₁ATPase in a molar ratio 1:1 rapidly reacts with H₂O₂ mediating a 30% enzyme inactivation. These results support the presence in mitochon-drial and thermophilic F₁ATPase of a conserved site involved in iron binding and in oxidative inactivation, in which iron exhibits similar redox properties. On the other hand, at variance with thermophilic F₁ATPase, the mitochondrial enzyme has the possibility of maintaining one equivalent of Fe(II) in its peculiar ATP-dependent site, besides one equivalent of Fe(III) in the conserved nucleotide-independent site. In this case mitochondrial F, ATPase undergoes a higher inactivation (75%) upon exposure to H₂O₂. Under all conditions the inactivation is significantly prevented by PBN and DMSO but not by Cu, Zn superoxide dis-mutase, thus suggesting the formation of OH radicals as mediators of the oxidative damage. No dityrosines, carbonyls or oxidized thiols are formed. In addition, in any cases no protein fragmentation or aggregation is observed upon the treatment with H₂O₂.     

Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller

Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO₃⁻ or NH₄⁺) present also influenced P. minimum response to DOC; 10–20 μg ml⁻¹ additions of fulvic acid had no effect upon growth rates in the presence of NH₄⁺ but significantly increased growth rates in the presence of NO₃⁻, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO₃⁻ assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O₂ cell⁻¹ h⁻¹, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml⁻¹. Low concentrations (5–10 μg ml⁻¹) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml⁻¹ resulted in a 30% decrease in O₂ evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O₂ cell⁻¹ h⁻¹ and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.     

The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay

We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H₂O₂), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5–9 of the p53 gene (codons 126, 153/54, 189 and the 3′ splice site of exon 9) were analysed by the RSM assay for H₂O₂-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H₂O₂-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H₂O₂ exposure, possibly implicating the 5OHdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues.     

Occurrence of okadaic acid in the feeding grounds of dugongs (Dugong dugon) and green turtles (Chelonia mydas) in Moreton Bay, Australia

Okadaic acid (OA) is a diarrhetic shellfish poison (DSP) produced by a number of marine organisms including the benthic dinoflagellate Prorocentrum lima, which are often found on seagrass. As seagrass forms the basis of the diet of dugong (Dugong dugon) and green turtle (Chelonia mydas), these herbivores may potentially be exposed to OA through ingestion of P. lima found on the seagrass. In this study, the abundance of epiphytic P. lima, on seagrass, and the concentration of OA produced by these epiphytic dinoflagellates was measured in Moreton Bay, Queensland, Australia. P. lima and OA were found on all four species of seagrass collected. OA was detected in epiphytic material collected from seagrass, with a maximum of 460 ng OA/kg_(wwtSG) found on Halophila spinulosa. From this information, the estimated maximum daily intake (DI) of OA by an adult dugong consuming 40 kg_(wwtSG)/day was 18,400 ng/day, and an adult turtle consuming 2 kg_(wwtSG)/day was 920 ng/day. Analysis by HPLC/MS/MS of 54 stranded dugongs and 19 stranded turtles did not yield OA above the detection limit of 10,000 ng/kg_{(animal tissue)}. OA was found on seagrass, however it was not detected in the tissue samples of dugongs and turtles.     

Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S) heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120

Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-I ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH. Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH. The corresponding domain of PetF contained acidic or nonpolar residues instead. To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli. In addition to the point mutants, two chimeric proteins, FdxH : PetF and PetF : FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other. Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase. This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena. In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP⁺ oxidoreductase (FNR) and photosystem I. The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP⁺ photoreduction.     

Kinetics and mechanism of the reduction of (ImH)[<Emphasis Type="Italic">trans</Emphasis>-RuCl<Subscript>4</Subscript>(dmso)(Im)] by ascorbic acid in acidic aqueous solution

A systematic study of the reduction of (ImH)[trans-RuCl₄(dmso)(Im)] (NAMI-A; dmso is dimethyl sulfoxide, Im is imidazole), a promising antimetastasing agent entering phase II clinical trial, by l-ascorbic acid is reported. The rapid reduction of trans-[Ru^IIICl₄(dmso)(Im)]⁻ results in formation of trans-[Ru^IICl₄(dmso)(Im)]²⁻ in acidic medium (pH = 5.0) and is followed by successive dissociation of the chloride ligands, which cannot be suppressed even in the presence of a large excess of chloride ions. The reduction of NAMI-A strongly depends on pH and is accelerated on increasing the pH. Over the small pH range 4.9−5.1, the reaction is quite pH-independent and the influence of temperature and pressure on the reaction could be studied. On the basis of the reported activation parameters and other experimental data, it is suggested that the redox process follows an outer-sphere electron transfer mechanism. A small contribution from a parallel reaction ascribed to inner-sphere reduction of aqua derivatives of NAMI-A, was found to be favored by lower concentrations of the NAMI-A complex and higher temperature. In the absence of an excess of chloride ions, the reduction process is catalyzed by the Ru(II) products being formed. The reduction of NAMI-A is also catalyzed by Cu(II) ions and the apparent catalytic rate constant was found to be 1.5 × 10⁶ M⁻² s⁻¹ at 25 °C. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).