Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS.

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 microl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 microl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 +/- 1.1 pmol, which was in complete agreement with the observed X(0) = 15.0 +/- 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.

Chemical aromatization of 19-hydroxyandrosta-1,4-diene-3,17-dione with acid or alkaline: elimination of the 19-hydroxymethyl group as formaldehyde.

In order to determine whether or not a 19-hydroxymethyl group of 19-hydroxyandrosta-1,4-diene-3,17-dione (2, 19-hydroxy ADD), an intermediate of aromatase-catalyzed estrone formation from ADD, a suicide substrate of aromatase, is eliminated as formaldehyde, we examine chemical nature of removal of the 19-hydroxymethyl group. 19-acetate 3 and 19-tert-butyldimethylsiloxy compound 4 are known to convert rapidly to estrone with treatment of NaOH or n-Bu4NF. Since compound 2 was unstable and unobtainable under these conditions, compounds 3 and 4 as equivalents to compound 2 were used in this study. The acetate 3 with 5 mol/l HCl in acetone and 10% KOH in MeOH along with the silyl ether 4 with 5 mol/l HCl in acetone and 1 mol/l n-Bu4NF in THF gave formaldehyde and estrone in which a ratio of the aldehyde to estrone was near 1. This result indicates that the 19-hydroxymethyl groups of compound 3 and 4 are eliminated as formaldehyde along with estrone derived from the steroid skeleton under the acid or base treatment. The findings suggest that a single hydroxylation at the 19 carbon of ADD (1) would be, chemically, all that was required for estrone formation.

Development of sensitive derivatization method for aldosterone in liquid chromatography-electrospray ionization tandem mass spectrometry of corticosteroids.

A highly sensitive quantification method of aldosterone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using recently developed picolinyl derivatization. Aldosterone was smoothly and quantitatively converted to the ethyl ether-picolinyl derivative by treatment with HCl-ethanol followed by the esterification with picolinic acid in the presence of 2-methyl-6-nitrobenzoic anhydride and 4-dimethylaminopyridine. The positive ion-ESI mass spectrum of the ethyl ether-picolinyl derivative was characterized by an appearance of protonated molecule ([M+H](+)) as a base peak. The ethyl ether-picolinyl derivatization gave a successful result in a separation of aldosterone from corticosterone, dehydrocorticosterone and cortexolone, and also provided an approximately 10-fold higher ESI response in the positive-LC-ESI-MS/MS (selected reaction monitoring; SRM) when compared to that of underivatized molecule (negative mode). The limit of quantification of aldosterone by SRM using ethyl ether-picolinyl derivatization (m/z 494-->m/z 448) was 1 pg/0.2 ml serum with accuracy and precision of 92.6% and 5.6%, respectively.

Mechanistic aspects of rearrangement of 16alpha-hydroxy-17-keto steroids to the 17beta-hydroxy-16-keto isomers.

The mechanistic aspects of the alkali-catalyzed rearrangement of 16alpha-hydroxy-17-keto steroid 1 to 17beta-hydroxy-16-keto steroid 2 are elucidated by use of (18)O- and deuterium-labeling experiments. The (18)O-labeling experiments refute the gem-hydration-quasi-diaxial dehydration mechanism for the rearrangement previously proposed and support the conventional enolization mechanism. Moreover, equilibrium by gem-hydration-dehydration occurs at the C-17 carbonyl more efficiently than at the C-16 carbonyl. Enolization rate of a carbonyl group at C-16 of 17beta-ketol 2 toward the C-17 position (k(16,17)) was about 8-10 times higher than those of 16alpha-ketol 1 toward the C-16 position (k(17,16)) and ketol 2 toward the C-15 position (k(16,15)). The marked deuterium-isotope effect on each enolization was observed with k(H)/k(D) ranging between 5.4 and 8.8. The present findings reveal that the initial hydration-dehydration equilibration at the C-17 carbonyl of ketol 1 followed by enolization of the carbonyl gives the ene-diol intermediate that isomerizes quantitatively to the 16-keto isomer of which the 16-carbonyl moiety enolizes preferentially toward the C-17 position rather than the C-15 position, yielding the ene-diol. Computational calculations of ground state energies of ketols 1-M and 2-M, trans-cyclohexane/cyclopentane structures, and their activation energies in the rearrangement support the dynamic aspects of the rearrangement as well as the kinetics data of the enolization.

Simultaneous determination of tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry.

Simultaneous quantification method of three major metabolites of cortisone and cortisol, tetrahydrocortisol, allotetrahydrocortisol and tetrahydrocortisone by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was investigated in a positive mode using a recently developed picolinyl derivatization. Conversion of each steroid into the corresponding picolinyl derivatives (1b, 2b or 3b) was performed by mixed anhydride method using picolinic acids and 2-methyl-6-nitrobenzoic anhydride. Derivatization proceeded smoothly to afford the corresponding 3, 21-dipicolinyl derivatives. Positive ion-ESI mass spectra of the picolinyl derivatives were dominated by an appearance of [M+H](+) as base peaks in all cases. The picolinyl derivatives provided 15 to 80-fold higher ESI response in the LC-ESI-MS/MS (selected reaction monitoring: SRM) when compared to those of underivatized molecules in a positive LC-ESI mode. The use of the picolinyl ester, solid-phase extraction, and deuterium labeled internal standards enabled the concentrations of these metabolites in human urine to be determined simultaneously by LC-ESI-MS/MS (SRM) with a small sample volume of less than 1microl urine.

Aromatization of androstenedione and 16alpha-hydroxyandrostenedione in human placental microsomes. Kinetic analysis of inhibition by the 19-oxygenated and 3-deoxy analogs.

Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively. 3-carbonyl and 16alpha-hydroxy functions of 16-OHAD play a critical role of selection of the 16-OHAD binding site. The results suggest that the AD derivatives 1 are kinetically aromatized at a different site from the 16-OHAD derivatives 2. Physical and/or chemical environments around the aromatase protein in the microsomal membrane may play a significant role in the expression of the substrate specificity, and the present results do not exclude the idea that the placental microsomes have a single binding site.

Proton Affinitive Derivatization for Highly Sensitive Determination of Testosterone and Dihydrotestosterone in Saliva Samples by LC-ESI-MS/MS.

In this study, proton affinitive derivatization using picolinic acid and its analogs (3- and 6-methylpicolinic acid and 5-butylpicolinic acid) with proton affinitive moieties was performed for the highly sensitive determination of testosterone (T) and 5α-dihydrotestosterone (DHT) in saliva by LC-ESI-MS/MS. T and DHT were converted to their corresponding picolinate esters and their chromatographic behavior was investigated with a reversed phase column. The picolinate ester of each steroid exhibited a clear single peak and elution occurred in the following order: picolinate, 3/6-methylpicolinate, and 5-butylpicolinate. Estimation and understanding of the separation and retention time of each picolinate ester was made simple using the develop method. Although the peaks of picolinate and 3/6-methylpicolinate esters were suppressed by interference from the saliva background (matrix effect), the 5-butylpicolinate esters were only marginally affected.

Mass spectrometric analysis of oxygenations in aromatization of androst-4-ene-3,6,17-trione, a suicide substrate of aromatase, by placental microsomes. Isotope effect and stereochemistry.

Aromatase catalyzes the conversion of androstenedione (AD) to estrone through three sequential oxygenations of the 19-methyl group. 6-OxoAD (1) is one of the typical suicide substrates of aromatase, which is converted by aromatase to 6-oxoestrone through 19-alcohol (19-ol) and 19-aldehyde (19-al) intermediates 2 and 3. To study the deuterium isotope effect on the conversion of 19-ol 2 to 19-al 3 as well as the stereochemistry of the 19-hydrogen removal in this conversion, we initially synthesized [19,19-(2)H(2)] and [19S- or 19R-(2)H] 19-ols 2, starting from the corresponding deuterium-labeled 19-hydroxyAD derivatives. In incubation of non-labeled and [19,19-(2)H(2)]-labeled 19-ol 2 or that of their 1:1 mixture with human placental microsomes in the presence of NADPH under air, there was no significant deuterium-isotope effect on the production of the aromatized product 6-oxoestrone or on the conversion of 19-ol 2 to 19-al 3, based on gas chromatography-mass spectrometric analysis of the estrogen product or liquid chromatography-mass spectrometric (LC-MS) analysis of the deuterium contents of the product 19-al 3 and the recovered 19-ol 2. Moreover, in the incubations of [19S-(2)H] 19-ol 2 and its 19R isomer, LC-MS analysis of the product 3 demonstrated that the 19-pro-R hydrogen atom was stereospecifically removed in the conversion of 19-ol 2 to 19-al 3. These findings indicate that the 19-oxygenation of 19-ol 2 would proceed in the same mechanism as that involved in the AD aromatization.

Use of novel picolinoyl derivatization for simultaneous quantification of six corticosteroids by liquid chromatography-electrospray ionization tandem mass spectrometry.

Simultaneous quantification method of six corticosteroids, cortisone, cortisol, cortexolone, corticosterone, dehydrocorticosterone and deoxycorticosterone, by LC-electrospray ionization (ESI)-MS in a positive mode using novel picolinoyl derivatization was investigated. Conversion of each corticosteroid into the corresponding picolinoyl derivative was performed by mixed anhydride method using picolinic acids and 2-methyl-6-nitrobenzoic anhydride. Derivatization proceeded smoothly to afford the corresponding 21-monopicolinoyl derivatives. Positive ion-ESI mass spectra of the picolinoyl derivatives were dominated by the appearance of [M+H](+) as base peaks. The picolinoyl derivatives provided 5-10 times higher ESI response in the LC-ESI-MS-selected reaction monitoring (SRM) when compared to those of underivatized molecules in a positive LC-ESI-MS mode. The use of the picolinoyl ester, solid-phase extraction, and deuterium labeled internal standards enabled to determine the concentrations of these corticosteroids in human saliva simultaneously by LC-ESI-MS-SRM.

Synthesis of pyridine-carboxylate derivatives of hydroxysteroids for liquid chromatography-electrospray ionization-mass spectrometry.

Synthesis and liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) behaviors of the picolinoyl, 6-methylpicolinoyl, nicotinoyl, 2-methoxynicotinoyl and isonicotinoyl derivatives of the hydroxysteroids estrone, estradiol, 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) and testosterone in positive mode were investigated. Each steroid was converted to the corresponding pyridine-carboxylate derivative by the acyl chloride method or the mixed anhydride method using the corresponding free acids and 2-methyl-6-nitrobenzoic anhydride; in each case, the latter method principally gave a better yield. The pyridine-carboxylate derivative of each steroid exhibited a clear single peak in liquid chromatography with a reversed phase column and CH(3)CN-0.1% CH(3)COOH as a mobile phase. The positive-ESI-mass spectra of the picolinoyl, 6-methylpicolinoyl and 2-methoxynicotinoyl derivatives showed a predominance of [M+H](+), whereas [M+H+CH(3)CN](+) was observed with high intensity in the nicotinoyl and isonicotinoyl derivatives. Even in the case of estradiol, with its two hydroxyl groups, a single charged ion of [M+H](+) or [M+H+CH(3)CN](+) was observed in the positive-ESI-mass spectrum of each derivative. The results revealed that picolinoyl derivatization is a simple and versatile method suitable for the sensitive and specific determination of hydroxysteroids by LC-ESI-MS (selected reaction monitoring).

Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry.

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.

Determination of testosterone concentrations in rat plasma using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry combined with ethyl oxime and acetyl ester derivatization.

A quantitative method for measuring testosterone (T) concentrations in rat plasma was developed using ethyl oxime and acetyl ester derivatization and liquid chromatography-atmosphere pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS). The method utilizes a solid phase extraction with Varian Bond Elut C18, a derivatization process to form testosterone ethoxime acetate and LC-APCI-MS/MS with a reversed phase LC and a C8 column. This method is capable of detecting testosterone concentrations as low as 0.2 ng/ml in a 0.05 ml sample of rat plasma. This method can be used as a sensitive chromatography-based assay for small sample volumes of rat blood.

Recent development of chemical derivatization in LC-MS for biomedical approaches.

LC-MS/MS is currently the most powerful system in biomedical analysis. At the same time, chemical derivatization is a useful technique to enhance the detection sensitivity of nonionizable or poorly ionizable molecules in LC-MS/MS. Derivatization improves the ionization efficiency, the chromatographic separation and/or the chemical stability. This article presents an overview of the recent development of chemical derivatization reagents and reactions for the quantitative analysis of xenobiotic and endogenous molecules such as pharmaceuticals, amino acids, peptides, proteins, steroids, biomarkers and industrial products by LC-MS.

Simultaneous quantification of salivary 3-hydroxybutyrate, 3-hydroxyisobutyrate, 3-hydroxy-3-methylbutyrate, and 2-hydroxybutyrate as possible markers of amino acid and fatty acid catabolic pathways by LC-ESI-MS/MS.

We have developed a highly sensitive and specific method for quantification of salivary 3-hydroxybutyrate (3HB), 3-hydroxyisobutyrate (3HIB), 3-hydroxy-3-methylbutyrate (3HMB) and 2-hydroxybutyrate (2HB), which could be new non-invasive biomarkers for catabolic pathways of fatty acids/ketogenic amino acids, valine, leucine, and methionine/threonine/α-ketobutyrate, respectively. The four hydroxybutyrates (3HB, 3HIB, 3HMB, and 2HB) were extracted from 5 µl of saliva, converted to 2-pyridylmethyl (2PM) ester derivatives, and measured by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. [(13)C4]3HB was used as an internal standard. The detection limits for the 2PM esters were <1 pg (7.9-9.6 fmol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of the hydroxybutyrates were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements were calculated to be 0.45-5.28 and 0.54-3.45 %, respectively. Experiments performed using 5 µl of saliva spiked with 3.8-154.4 pmol of the four hydroxybutyrates gave recoveries of 98.5 to 108.8 %, with a mean recovery of 104.1 %. In vitro experiments in hepatocytes or skeletal muscle cells showed that addition of palmitic acid, valine, leucine or α-ketobutyrate to culture medium markedly increased the targeted hydroxybutyrate concentrations. The salivary concentration of each targeted hydroxybutyrate was positively correlated with that in serum, and the salivary levels were elevated in patients with liver cirrhosis, which is characterized by upregulated catabolism of lipids and amino acids. The proposed method is useful for quantification of salivary 3HB, 3HIB, 3HMB, and 2HB for monitoring of catabolic activities of amino acids and fatty acids.

Measurement of peripheral plasma 18-oxocortisol can discriminate unilateral adenoma from bilateral diseases in patients with primary aldosteronism.

Adrenal venous sampling is currently the only reliable method to distinguish unilateral from bilateral diseases in primary aldosteronism. In this study, we attempted to determine whether peripheral plasma levels of 18-oxocortisol (18oxoF) and 18-hydroxycortisol could contribute to the clinical differentiation between aldosteronoma and bilateral hyperaldosteronism in 234 patients with primary aldosteronism, including computed tomography (CT)-detectable aldosteronoma (n=113) and bilateral hyperaldosteronism (n=121), all of whom underwent CT and adrenal venous sampling. All aldosteronomas were surgically resected and the accuracy of diagnosis was clinically and histopathologically confirmed. 18oxoF and 18-hydroxycortisol were measured using liquid chromatography tandem mass spectrometry. Receiver operating characteristic analysis of 18oxoF discrimination of adenoma from hyperplasia demonstrated sensitivity/specificity of 0.83/0.99 at a cut-off value of 4.7 ng/dL, compared with that based on 18-hydroxycortisol (sensitivity/specificity: 0.62/0.96). 18oxoF levels above 6.1 ng/dL or of aldosterone >32.7 ng/dL were found in 95 of 113 patients with aldosteronoma (84%) but in none of 121 bilateral hyperaldosteronism, 30 of whom harbored CT-detectable unilateral nonfunctioning nodules in their adrenals. In addition, 18oxoF levels below 1.2 ng/dL, the lowest in aldosteronoma, were found 52 of the 121 (43%) patients with bilateral hyperaldosteronism. Further analysis of 27 patients with CT-undetectable micro aldosteronomas revealed that 8 of these 27 patients had CT-detectable contralateral adrenal nodules, the highest values of 18oxoF and aldosterone were 4.8 and 24.5 ng/dL, respectively, both below their cut-off levels indicated above. The peripheral plasma 18oxoF concentrations served not only to differentiate aldosteronoma but also could serve to avoid unnecessary surgery for nonfunctioning adrenocortical nodules concurrent with hyperplasia or microadenoma.

An innovative LC-MS/MS-based method for determining CYP 17 and CYP 19 activity in the adipose tissue of pre- and postmenopausal and ovariectomized women using 13C-labeled steroid substrates.

CONTEXT: Does adipose tissue produce steroid hormones like an endocrine organ? OBJECT: To clarify whether adipose tissue produces sex steroid hormone like an endocrine organ, we estimated several key steroid hormone levels, as well as CYP17 and CYP19 activity, in ovariectomized, pre- and postmenopausal women by liquid chromatography-tandem mass spectrometry (LC-MS/MS). SUBJECTS AND METHODS: The subjects were 19 premenopausal (n = 12), postmenopausal (n = 4), and ovariectomized women (n = 3) aged 27-68 years. Serum, visceral adipose and sc adipose samples were taken from these subjects and stored at -70°C. The levels of cortisol, cortisone, progesterone (Prog), androstenedione, dehydroepiandrosterone, estrone, estradiol (E2), and T in serum and adipose tissue were estimated simultaneously by LC-MS/MS. CYP17 and CYP19 activity in tissues were assayed with the use of (13)C-labeled steroid precursors and LC-MS/MS-based estimation of the metabolites. RESULTS: E2 and Prog levels in the sera of postmenopausal or ovariectomized women were less than 10% of those in premenopausal women. No marked variations were seen in other hormones. Estrone, androstenedione, dehydroepiandrosterone, and Prog levels in the visceral and sc tissues of postmenopausal and ovariectomized women were 9-60 times higher than those in serum, whereas E2 and T levels were 3- to 7-fold higher than those in serum, and cortisol and cortisone levels were 20% of those found for serum. CYP17 in adipose tissue was found to have 17-hydroxylase and 20,17-lyase activity, with each catalytic activity being essentially equal. Therefore, CYP17 in adipose tissue is of the testicular/ovarian type but not adrenal type, which has 17-hydroxylase activity dominant. The presence of CYP19 activity in adipose tissue was approximately 3% of CYP17. CONCLUSION: Our findings suggest that adipose tissue acts as an endocrine organ, with CYP17 and CYP19 activity playing an essential role in sex steroid hormone biosynthesis.

Structures and biomimetic synthesis of novel α-pyrone polyketides of an endophytic Penicillium sp. in Catharanthus roseus.

Novel polyketides, citreoviripyrone A (1) and B (2), known citreomontanin (3), and (-)-citreoviridin (4) were isolated from the mycelium of the endophytic fungus. The endophytic fungus, which belongs to the genus Penicillium, was separated from surface-sterilized healthy leaves of Catharanthus roseus. The structures of 1 and 2 were determined on the basis of NMR data, and 1 was characterized as an α-pyrone polyketide featuring bicyclo[4.2.0]octadiene. The biomimetic synthesis of 1 and 2 from 3 elucidated a plausible biosynthetic pathway. Both Zn(II)-type and NAD(+)-dependent histone deacetylase inhibitors significantly enhanced the production of 1 and 3.

Benzophenones from an endophytic fungus, Graphiopsis chlorocephala, from Paeonia lactiflora cultivated in the presence of an NAD+-dependent HDAC inhibitor.

Graphiopsis chlorocephala was separated from the surface-sterilized healthy leaves of Paeonia lactiflora (Paeoniaceae) and cultivated with nicotinamide (an NAD(+)-dependent HDAC inhibitor). The culture conditions significantly enhanced secondary metabolite production in the fungus and led to the isolation of a structurally diverse set of new benzophenones, cephalanones A-F (1-6), and a known 2-(2,6-dihydroxy-4-methylbenzoyl)-6-hydroxybenzoic acid (7). The structures of 1-6 were determined from NMR data, single crystal X-ray diffraction, and chemical transformations.

Tenuipyrone, a novel skeletal polyketide from the entomopathogenic fungus, Isaria tenuipes, cultivated in the presence of epigenetic modifiers.

The concomitant addition of the histone deacetylase inhibitor and the DNA methyltransferase inhibitor to the culture medium of an entomopathogenic fungus, Isaria tenuipes, greatly enhanced its secondary metabolite production and led to the isolation of tenuipyrone (1), a novel polyketide with an unprecedented tetracyclic ring system bearing a spiroketal structural component, along with two known C(10)-polyketides, cephalosporolide B (2), which is a plausible biosynthetic precursor of 1, and cephalosporolide F (3).