Carboxylic Acid Form Research Articles

Studies on the Metabolic Fate of a Novel Orally Active Nonpeptide Endothelin Antagonist TA-0201: Metabolism and First-pass Metabolism of TA-0201 in Rats.

TA-0201は非ペプチド性の新規エソドセリン受容体拮抗薬である．TA-0201のラットにおける代謝および体内動態について検討した．さらに，ヒトを含めた各動物種肝ミクロゾームを用いてin vitroにおける代謝について検討した． 1．TA-0201の主代謝はカルボソ酸体(CA)およびジオール体(Diol)への酸化反応であり，各動物種の肝ミクロゾームにおけるTA-0201からCAへの代謝固有クリアランス(in vitro)はサル>>イヌ>ヒト>ラットの順で大きいことが判明した．また，TA-0201からCAおよびジオール体への代謝反応にはCYP3A Familyが主に関与していることが推定された． 2．TA-0201のラットにおける血漿中動態を薬物速度論的に解析したところ，経口および静脈内投与時の血漿中濃度推移から求めたBAは約60%と良好であり，本化合物はラットの体内からほぼ90%以上が代謝により消失し，その代謝臓器は主に肝臓と消化管であることが示唆された．肝および消化管における臓器抽出率はそれぞれ0.21および0.11であり，両臓器における初回通過効果の寄与はほぼ同程度であると推定された．

Photodecarboxylation of DX-9065a, a new factor Xa inhibitor, in aqueous solution.

The photodegradation reaction of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3- (7-amidino-2-naphthyl) propanoic acid hydrochloride pentahydrate (DX-9065a), a new factor Xa inhibitor, was investigated in aqueous solution at various pH values (1.1-8.0) at 25 degrees C. The photodegradation of DX-9065a followed apparent first-order kinetics under artificial sunlight in a fairly good manner. The photodegradation rates of DX-9065a in a neutral solution were higher than those in acidic solution. The log k-pH profile indicated that the photodegradation rate of DX-9065a was related to the dissociation of the carboxyl group and suggested that the main photo-labile species was the carboxylate form. Further kinetic study showed that the carboxylate form of DX-9065a underwent decarboxylation quantitatively to produce D41-1077, the photodecarboxylation product, on irradiation, while the carboxylic acid form of DX-9065a did not perform any photodecarboxylation. Moreover, the photodecarboxylation mechanism was discussed in relation to the kinetics and photodegradation pathway.

The 2-Oxocyclopentanecarboxylic Acid Keto−Enol System in Aqueous Solution: Generation of the Enol by Hydration of an Acylketene

Flash photolysis of 2-diazocyclohexane-1,3-dione in aqueous solution produced 2-ketocyclopentylideneketene, which hydrated to the enol of 2-oxocyclopentanecarboxylic acid, and the enol then isomerized to the keto form of the acid. This ketene proved to be a remarkably reactive substance, with an uncatalyzed hydration rate constant of k = 1.4 × 106 s-1 and a hydroxide-ion-catalyzed rate constant of 7.5 × 107 M-1 s-1; no acid catalysis of hydration was found. Ketonization of the enol was a slower process with rate constants in the millisecond to second range. The reaction shows a complex rate profile that could be interpreted in terms of rate-determining proton transfer to the β-carbon atom of successively ionized forms of the enol in successively more basic solutions. In concentrated acid solutions, the carboxylic acid group of the enol also underwent equilibrium protonation of its carbonyl group, with pKSH = −3.86. Acidity constants of the carboxylic acid group of the enol, pQa,E = 4.16, and the hydroxyl group of the enol, pQaE = 12.41, were also determined. (These acidity constants are concentration quotients applicable at an ionic strength of 0.10 M.) Rates of enolization of the keto form of the substrate were also measured by bromine scavenging, and these, in combination with ketonization rates, gave the keto−enol equilibrium constants pKE = 2.51 for the system in the un-ionized carboxylic acid form and pK‘E = 3.00 for the ionized carboxylate form. The acidity constant of the carboxylic acid group of the keto form was determined as well: pQa,K = 3.67.

Inhibition of palmito ( Acanthophoenix rubra) polyphenol oxidase by carboxylic acids

The inhibition of palmito ( Acanthophoenix rubra) polyphenol oxidase (PPO) is reported. Recently, two forms of palmito PPO were partially purified by hydrophobic chromatography. Inhibitory effects of various carboxylic acids on these two forms have been studied. Both forms showed identical behaviour towards the inhibitors studied. Cinnamic acid was found to have the greatest inhibitory effect ( K i = 0·06 mM). When the inhibitory effects of acids from the benzoic acid family and from the cinnamic acid family were compared, it was found that acids which possess a double bond between the benzene ring and the carboxylic function showed the highest inhibitory effect. This inhibitory effect was decreased by substitutions on the benzene ring. The influence of pH on the inhibitory effect of carboxylic acids on palmito PPO has also been investigated. K i decreased with a decrease in pH. This effect is due to the fact that it is only the undissociated form (AH) of carboxylic acids that is responsible for inhibition of PPO. Inhibition constants ( K i for the AH form have been recalculated and were found to remain constant in the pH range studied (for benzoic acid K i = 0·14 mM, for cinnamic acid K i = 0·019 mM, for sorbic acid K i = 0·15 mM).

Binding of the growth factor glycyl- l-histidyl- l-lysine by heparin

Evidence is presented that the growth factor glycylhistidyl-lysine (GHK) binds to heparin, and the interaction has been characterized by [ 1H]NMR spectroscopy. 1H chemical shifts indicate that GHK interacts with both the carboxylic acid and the carboxylate forms of heparin. The chemical shift data are consistent with a weak delocalized binding of the triprotonated (ImH +, GlyNH 3 +, LysNH 3 +) form of GHK by the carboxylic acid form of heparin. As the pD is increased and the carboxylic acid groups are titrated, chemical shift data indicate that ammonium groups of GHK are hydrogen bonded to heparin carboxylate groups, while the histidyl imidazolium ring occupies the imidazolium-binding site of heparin. Evidence for site-specific binding includes displacement of chemical shift titration curves for heparin to lower pD, increased shielding of specific heparin protons by the imidazolium ring current and displacement of chemical shift titration curves for GHK to higher pD. Specific binding constants were determined for binding of the (ImH +, GlyNH 3 +), LysNH 3 +), (ImH +, GlyNH 2, LysNH 3 +) and (Im, GlyNH 3 +, LysNH 3 +) forms of GHK by the carboxylate form of heparin from chemical shift vs. pD titration data.

Multinuclear magnetic resonance studies of the interaction of inorganic cations with heparin

The interaction of Na +, Ca 2+, Mg 2+, Zn 2+ and La 3+ with heparin, a highly negatively charged glycosaminoglycan, was studied by 1H and 23Na nuclear magnetic resonance spectroscopy. 1H chemical shift and nuclear Overhauser effect (NOE) data indicate that the counter ions Na +, Ca 2+ and Mg 2+ interact with the low pH, carboxylic acid form of heparin by delocalized, long-range electrostatic interactions. At higher pH, 1H chemical shift and NOE data indicate that Na + and Mg 2+ continue to interact with heparin in the same manner, even upon deprotonation of the carboxylic acid group; however, there is a site-specific contribution to the binding of Ca 2+, Zn 2+ and La 3+ under these conditions. Acid dissociation constants for heparin carboxylic acid groups and heparin-metal binding constants were determined from the pH dependence of 1H chemical shifts and 23Na spin-lattice ( T 1) relaxation times. Equilibrium constants for exchange of M 2+ for heparin-bound Na + were obtained from 23Na T 1 data. The acid dissociation constants show a strong dependence on Na + concentration due to the polyelectrolyte character of heparin.

X-ray structure and catalytic mechanism of lobster enolase

Enolase prepared from lobster tail muscle yielded trigonal crystals with one 47 kDa subunit per asymmetric unit. X-ray data were collected on the apoenzyme at 2.4 A resolution and on a complex with Mn2+ and the inhibitor phosphoglycolate at 2.2 A resolution. The corresponding cDNA was amplified from a library of lobster muscle cDNA, and a sequence corresponding to residues 27-398 was determined. It is highly homologous to other enolases, including yeast enolase for which an X-ray structure is available. Yeast enolase was used as a starting point for crystallographic refinement, which led to models of lobster enolase having R-factors below 22% and good stereochemistry. These models are very similar to yeast enolase; they have the same fold with a beta 3 alpha 4 N-terminal domain followed by an atypical alpha/beta barrel. Lobster apoenolase and the ternary complex differ only in the position of three mobile loops. In the complex, a single Mn2+ ion is seen ligated to three carboxylates and three water molecules. Phosphoglycolate binds near, but not directly to, the metal. His 157, which belongs to one of the mobile loops, is in contact with the C2 atom of the ligand. A water molecule hydrogen-bonds to the carboxylate of the ligand and to those of Glu 166 and Glu 209. We suggest that His 157 is the base that abstracts the C2H proton, whereas the water molecule is part of a proton relay system keeping the substrate in the carboxylic acid form where the pKa of the C2H group is low enough for proton transfer to His 157. The resulting catalytic mechanism is different from those proposed on the basis of the yeast enzyme X-ray structures, but it fits with earlier biochemical and spectroscopic data.

The effect of the thirteen Dip-allatostatins on myogenic and induced contractions of the cockroach ( Diploptera punctata) hindgut

The Dip-allatostatins are a group of 13 neuropeptides, ranging in size from 6 to 18 amino acids, identified from a cloned cDNA sequence of Diploptera punctata. All 13 Dip-allatostatins inhibited both myogenic and proctolin-induced contractions of the hindgut of D. punctata. Dip-allatostatins were capable of reducing a 5 × 10 −6 M proctolin-induced contraction in a dose-dependent manner, with ED 50 values ranging from 1.7 × 10 −7 M for Dip-allatostatin 7 to greater than 1.3 × 10 −5 M for Dip-allatostatin 12. The effects of the Dip-allatostatins were reversible upon washing with saline. The free carboxylic acid (non-amidated) form of Dip-allatostatin 11, callatostatin 3 and callatostatin 5 showed a loss of inhibitory activity. In contrast to the inhibitory actions of the Dip-allatostatins on hindgut contractions, selected Dip-allatostatins (Dip-allatostatins 4, 6, 7, 10, 12), at concentrations ranging from 10 −7 M to 2 × 10 −5 M, did not alter the frequency or amplitude of myogenic contractions or inhibit proctolin-induced contractions of the cockroach oviduct muscle. Dip-allatostatins (Dip-allatostatins 1, 2, 4, 5, 6, 7) also had no effect on either myogenic or proctolin-induced contractions of the oviduct of Locusta migratoria.

Site-directed mutagenesis on (serine) carboxypeptidase Y. A hydrogen bond network stabilizes the transition state by interaction with the C-terminal carboxylate group of the substrate.

The three-dimensional structure of (serine) carboxypeptidase Y suggests that the side chains of Trp49, Asn51, Glu65, and Glu145 could be involved in the recognition of the C-terminal carboxylate group of peptide substrates. The mutations Trp49-->Phe; Asn51-->Ala, Asp, Glu, Gln, Ser, or Thr; Glu65-->Ala; and Glu145-->Ala, Asp, Asn, Gln, or Ser have been performed. Enzymes with Ala at these positions were also produced as double and triple mutations. These mutations have only little effect on the esterase activity of the enzyme, consistent with the absence of a hydrogen bond acceptor in the P1' position of such substrates. On the other hand, removal of the hydrogen-bonding capacity by incorporation of Ala at any of these four positions results in reduced peptidase activity, in particular when Asn51 and Glu145 are replaced. The results are consistent with Trp49 and Glu65 orienting Asn51 and Glu145 by hydrogen bonds, such that these can function as hydrogen bond donors (Glu145 only in its protonated carboxylic acid form) with the C-terminal alpha-carboxylate group of the peptide substrate as acceptor. However, it appears that strong interactions are formed only in the transition state since the combined removal of Asn51 and Glu145 reduces kcat about 100-fold and leaves KM practically unchanged. The results obtained with enzymes in which Asn51 or Glu145 has been replaced with other residues possessing the capacity to donate a hydrogen bond demonstrate that there is no flexibility with respect to the nature of the hydrogen bond donor at position 145, whereas enzymes with Gln, Ser, or Thr at position 51 exhibit much higher activity than N51A, although none of them reaches the wild-type level. With carboxypeptidase Y as well as other serine carboxypeptidases the binding of peptide substrates in the ground state (KM) is adversely affected by an increase in pH. It is shown that deprotonation of a single ionizable group with a pKa of 4.3 on the enzyme is responsible for this pH effect. The results show that the group involved is either Glu65 or Glu145, the latter being the more probable. The effect of this ionization on KM is explained by charge repulsion between the carboxylate group of the substrate and that of Glu145, hence preventing substrate from binding.

Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions.

In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).

Fatty acids in layered metal hydroxides: membrane-like structure and dynamics

Layered metal hydroxides consist of sheets of positively charged edge-sharing M(OH){sub 6} octahedra as exemplified by the hydrotalcite family of materials. Anions occupy positions in the interlayers. In this study, we focus on the incorporation of long-chain fatty acids (CH{sub 3}(CH{sub 2}){sub n}COOH, n = 10, 12, 14) in between these layers. These materials mimic membranes and exhibit interesting packing and dynamic characteristics. The packing is strongly influenced by the charge distribution of the metal hydroxide layer. In the case of LiAl{sub 2}(OH){sub 6}{sup +} (25 {angstrom}{sup 2}/unit charge), a monomolecular film is formed, whereas a bimolecular film is formed in Mg{sub 3}Al(OH){sub 8}{sup +} (33 {angstrom}{sup 2}/unit charge). In the lithium aluminum hydroxide, the carboxylic acid is ion-exchanged into the layers, but in magnesium aluminate it is intercalated as the carboxylic acid form. On the basis of diffraction, calorimetry, and vibration spectroscopy, the dynamics of the films as a function of temperature has been explored. For LiAl{sub 2}(OH){sub 6}{sup +}, the spacing between the layers increases with temperature and retains an all-trans packing of the alkyl chains. In the Mg{sub 3}Al(OH){sub 8}{sup +} sample, the spacing between the layers decreases with temperature and the bimolecular film becomes disordered throughmore » the formation of kinks and gauche blocks at temperatures exceeding 130 {degrees}C. The control of packing density and dynamics of the alkyl chains via the charge density of the metal hydroxide layer results in the formation of novel, membrane-like materials. 26 refs., 21 figs.« less

Retinoids inhibit phospholipase A2 in human synovial fluid and arachidonic acid release from rat peritoneal macrophages

Retinoids have demonstrated antiinflammatory activity in certain animal models and human disease states. The mechanism by which retinoids elicit this activity is unknown. Some retinoids are known to inhibit arachidonic acid (AA) release and metabolism in intact cells in vitro. Retinoids may exert their antiinflammatory effects by inhibiting phospholipase A2 (PLA2) and the resultant production of inflammatory AA metabolites. Retinoids were evaluated in vitro as inhibitors of the PLA2 activity in human synovial fluid (HSF-PLA2). Of the naturally occurring, nonaromatic retinoids tested, all-trans-retinal, all-trans-retinoic acid (all-trans-RA) and 13-cis-RA were the most potent inhibitors (IC50 S 6-15 microM), whereas all-trans-retinol was much less potent. Of the synthetic aromatic retinoids and arotinoids examined, the free carboxylic, sulfonic, and sulfinic acid forms were more than 15-fold more potent inhibitors of HSF-PLA2 than their corresponding ethyl esters. These retinoids also were evaluated as inhibitors of calcium ionophore A23187-induced AA release from rat peritoneal macrophages. All-trans-RA and 13-cis-RA were potent inhibitors of AA release from these cells (IC50 S 4 microM), while the other natural retinoids were inactive. Of the aromatic retinoids and arotinoids tested, the free acid forms (IC50 S 2-6 microM) were 5- to 21-fold more potent inhibitors of AA release from the macrophages than their corresponding ethyl esters. The potencies of the arotinoids as inhibitors of HSF-PLA2 appeared to correlate with their potencies as inhibitors of AA release from A23187-stimulated rat peritoneal macrophages. These data support the hypothesis that one possible mechanism for the known antiinflammatory activity of some retinoids may be by inhibition of phospholipase A2.

Pharmacokinetic and tissue residue characteristics of fenprostalene, a prostaglandin F2α analog, in swine

SUMMARY Fenprostalene, a prostaglandin F2α analog, can be used to induce parturition in swine. As part of the approval process for that indication, pharmacokinetic characteristics of the absorption and elimination of fenprostalene and the depletion of drug residues from the principal edible tissues of swine were studied. Blood samples, urine, and feces were collected from 8 gilts (body weight, 95 ± 1.7 kg) for up to 72 hours after a single dose of 0.5 mg of 13,14-[3H]-fenprostalene in polyethylene glycol-400 was administered sc. At intervals of 24, 48, 72, and 168 hours after dosing, 2 gilts each were killed, and samples of liver, kidney, muscle, and abdominal fat were obtained for analysis. The mean (± sem) maximal concentration of fenprostalene radioequivalents in plasma (0.41 ± 0.05 nanogram-equivalents/ml; n = 8) was observed at 12 hours and decreased biexponentially, with half-lives of approximately 8 hours and 9 days. Mean cumulative recovery (n = 4) of the administered dose by 72 hours was 61.2 ± 5.9% in urine and 18.5 ± 2.6% in feces. The highest tissue fenprostalene concentration was in kidneys and liver, probably reflecting the role of those organs in excreting fenprostalene. Rates of depletion of fenprostalene equivalents from the injection site, kidneys, and liver were comparable with those previously observed in cattle. The composition of residue in the liver of 2 gilts slaughtered 12 hours after sc administration of [3H]-fenprostalene was examined in a second study. Results suggested that approximately 4% of the total residue was pharmacologically potent fenprostalene or the carboxylic acid form of fenprostalene. Approximately 29% of the residue was extensively degraded to acidic metabolites. The remaining 67% was bound, nonextractable material.

Acidic Permanganate Oxidations of Lignin and Model Compounds: Comparison with Ozonolysis

Acid permanganate oxidation degrades completely the aromatic moieties of lignins and lignin model compounds while a part of the side-chain structures can be recovered intact in the form of carboxylic acids. Thus, threonic acid (6.9%) is obtained from threo-veratrylglycerol, erythronic acid (7.8%) from erythro-veratrylglycerol φ-(2-methoxyphenyl) ether and glycolic acid (∼13%) from vanillyl alcohol. Erythronic and threonic acids, in a molar ratio of 0.6:1, were also identified as permanganate oxidation products of black spruce native lignin. Minor oxidation products from lignin and lignin model compounds included glyceric, oxalic, glycolic, tartronic, erythraric, threaric, 2,4-dihydroxybutanoic, succinic and malic acids

Soybean lipoxygenase-1 enzymically forms both (9 S)- and (13 S)-hydroperoxides from linoleic acid by a pH-dependent mechanism

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13 S)-(9 Z,11 E)-13-hydroperoxy-9,11-octadecadienoic acid and (9 S)-(10 E,12 Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5. As the pH decreased below 8.5, the proportion of (9 S)-hydroperoxide increased linearly until at pH 6 it constituted about 25% of the chiral products attributed to enzymic action. Below pH 6, lipoxygenase activity was barely measurable, and the hydroperoxide product arose mainly from autoxidation and possibly non-enzymic oxygenation of the pentadienyl radical formed by the enzyme. The change in percent enzymically formed 9-hydroperoxide between pH 6.0 and 8.5 paralleled the pH plot of a sodium linoleate/linoleic acid titration. It was concluded that the (9 S)-hydroperoxide is formed only from the nonionized carboxylic acid form of linoleic acid. Methyl esterification of linoleic acid blocked the formation of the (9 S)-hydroperoxide by lipoxygenase-1, but not the (13 S)-hydroperoxide. Since the hydroperoxydiene moieties of the (9 S)- and (13 S)-hydroperoxides are spatially identical when the molecules are arranged head to tail in opposite orientations, it is suggested that the carboxylic acid form of the substrate can arrange itself at the active site in either orientation, but the carboxylate anion can be positioned only in one orientation. These observations, as well as others in the literature, suggest an active-site model for soybean lipoxygenase-1.

ChemInform Abstract: The Hydrolytic Reactivity of Homoprostacyclin: Implications for the Physiological Control of Bleeding.

AbstractIn contrast to prostacyclin (Ia) (lifetime at physiological pH only 3 min) its homolog (Ib) (lifetime 30 min) is only 8 times more reactive in its carboxylate than in its carboxylic acid form (as a result of intramolecular catalysis), whereas prostacyclin (Ia) itself has a 100‐fold increase in reactivity.

The hydrolytic reactivity of homoprostacyclin: implications for the physiological control of bleeding

Rates of hydrolysis of the vinyl ether group of homoprostacyclin (2), which differs from prostacyclin only in that it has four instead of three carbon atoms in the chain joining this group to a carboxy function, were measured in H2O solution over the pH range 1–9 and in D2O solution in D2PO4––DPO42– buffers. The rate profile so obtained shows that homoprostacyclin is eight times more reactive in its carboxylate than in its carboxylic acid form, and an isotope effect on the accelerated reaction indicates that this rate increase is the result of intramolecular catalysis by the carboxylic acid group. This eight-fold acceleration is significantly less than the 100-fold increase produced by intramolecular catalysis in the case of prostacyclin itself, and that suggests that natural evolution has given prostacyclin the correct lifetime it needs to be effective in the physiological mechanism for the control of bleeding by adjusting the length of the carbon chain joining its vinyl ether and carboxy groups.

High Resolution Solid State 13C NMR Spectroscopy of Sporopollenins from Different Plant Taxa

The extremely chemically resistant component of the cell wall of spores, pollens, and some microorganisms, sporopollenin, is generally accepted to be derived from carotenoids or carotenoid esters. However, we report here that (13)C NMR analyses of sporopollenin from several sources shows that this widely held view is incorrect, with one possible exception. Sporopollenin is not a unique substance but rather a series of related biopolymers derived from largely saturated precursors such as fatty acids. The biopolymers contain widely varying amounts of oxygen in the form of ether, hydroxyl, carboxylic acid, ester, and ketone groups.

Evaluation of retinoids as inhibitors of [3H] all-trans retinoic acid binding to cellular retinoic acid-binding protein in rat skin and testes.

These experiments were designed to test the ability of certain analogs and metabolites of all-trans-retinoic acid (RA), 13-cis-retinoic acid, 4-hydroxy-all-trans-retinoic acid, 4-keto-all-trans-retinoic acid, trimethylmethoxyphenol (TMMP) analog of retinoic acid, and TMMP analog of ethyl retinoate (etretinate) to compete for cellular retinoic acid-binding protein (CRABP) in skin and testes of rats. All retinoids, except etretinate, bind to CRABP in a competitive manner with a similar affinity (approximately 5 X 10(-9) M for skin and 3 X 10(-9) M for testes). In contrast, etretinate binds in a noncompetitive manner with a much lower affinity (7.7 X 10(-5) M for skin and 7.5 X 10(-5) M for testes). The values (microM) of IC50 for CRABP from rat skin are 0.43, 0.41, 0.95, 0.83, and 77.4 and those from rat testes are 0.59, 1.29, 2.25, 2.30, and 75.25 for all-trans-RA, 13-cis-RA, 4-hydroxy-all-trans RA, 4-keto-all-trans-RA, TMMP analog of RA, and etretinate, respectively. Etretinate is a potent retinoid that is used in the treatment of psoriasis. The lack of quantitative correlation between IC50 and the biological activity of etretinate may be explained in that the active form of etretinate in the body may be the carboxylic acid form (TMMP analog of RA) which binds to CRABP with higher affinity.

The formation of a novel free radical metabolite from CCl4 in the perfused rat liver and in vivo.

Electron spin resonance spectroscopy has been used to monitor free radicals formed during CCl4 metabolism by perfused livers from phenobarbital-treated rats. Livers were perfused simultaneously with the spin trap phenyl N-t-butylnitrone and with either 12CCl4 or 13CCl4. Perfusate samples and CHCl3:CH3OH extracts of perfusate and liver samples were analyzed for phenyl N-t-butylnitrone radical adducts of reactive free radicals. In the organic extracts, hyperfine coupling constants and 13C isotope effects observed in the ESR spectra indicated the presence of the radical adduct of the trichloromethyl radical. Surprisingly, an additional free radical signal about two orders of magnitude more intense than that of the phenyl N-t-butylnitrone/CCl.3 radical adduct was observed in the aqueous liver perfusate. This adduct was also detected by ESR in rat urine 2 h after intragastric addition of spin trap and CCl4. This radical adduct had hyperfine coupling constants and 13C isotope effects identical with the radical adduct of the carbon dioxide anion radical (CO2-.). Analysis of the pH dependence of the coupling constants yielded a pK alpha of 2.8 for the CO2-. radical adduct formed either in the perfused liver or chemically. Carbon tetrachloride is converted into CCl.3 by cytochrome P-450 through a reductive dehalogenation. The trichloromethyl free radical reacts with oxygen to form the trichloromethyl peroxyl radical, CCl3OO., which may be converted into .COCl and then trapped. This radical adduct would hydrolyze to the carboxylic acid form, which is detected spectroscopically. Alternatively, the carbon dioxide anion free radical could form through complete dechlorination and then react with the spin trap to give the CO2-. radical adduct directly.