Ether lipid metabolism. Incorporation of O-hexadecyl ethanediol into rat brain lipids.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasmalogen analogue. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.     

Biosynthesis of O-acyl ethanediol phosphorylcholine in a cell-free system from rat liver.

1-$\text{0}$-Hexadecanoyl [U-¹⁴C]ethanediol can serve as substrate in the formation of 1-$\text{0}$-hexadecanoyl ethanediol 2-phosphorylcholine by particulate cell-free preparations from rat liver. Catalytic activity is largely associated with the microsomal fraction. The reaction requires CDPcholine and Mg⁺⁺. Phosphatidylcholine cannot substitute for CDPcholine, but Mn⁺⁺ is almost as effective as Mg⁺⁺. Ca⁺⁺ inhibits the reaction. The acyl ethanediol phosphorylcholine produced was identified by repeated cochromotography with authentic diol phospholipid to constant specific radioactivity, and by enzymatic and chemical degradations.     

On the organ specificity of neutral glycerolester hydrolase of various tissues; an immunological study

Rat liver microsomal glycerol monoester hydrolase (EC 3.1.1.23) has been purified 130 fold. The enzyme has a molecular weight of about 60,000. An antibody raised against this enzyme in rabbit did not inhibit heparin-releasable liver lipase, which hydrolyses long-chain 1- and 2-monoglycerides effectively. This confirms an earlier conclusion, based on results obtained with an antibody raised against the latter enzyme, that the non-releasable and heparin-releasable liver enzymes are different proteins. The antibody against the liver microsomal glycerol monoester hydrolase, however, inhibited also the monoglyceridase activities of acetone powder extracts of rat small intestinal epithelial microsomes and rat epididymal fat pads, suggesting structural similarities between the endoplasmic reticulum hydrolases of various tissues. These findings also apply to pig where an antibody against adipose tissue lipases inhibits the monoglyceridase activities of small intestinal and liver microsomal acetone powder extracts.     

Monoacylglycerol lipase activity in cardiac myocytes

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Abstract: Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg²⁺ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.     

Differences between microsomal and mitochondrial-matrix palmitoyl-coenzyme A hydrolase, and palmitoyl-L-carnitine hydrolase from rat liver.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) and palmitoyl-L-carnitine hydrolase (EC 3.1.1.28) activities from rat liver were investigated. 1. Microsomal and mitochondrial-matrix palmitoyl-CoA hydrolase activities had similar pH and temperature optima, although the activities showed different temperature stability. They were inhibited by Pb2+ and Zn2+. The palmitoyl-CoA hydrolase activities in microsomal fraction and mitochondrial matrix were differently affected by the addition of Mg2+, Ca2+, Co2+, K+ and Na+ to the reaction mixture. ATP, ADP and NAD+ stimulated the microsomal activity and inhibited the mitochondrial-matrix enzyme. The activity of both the microsomal and mitochondrial-matrix hydrolase enzymes was specific for long-chain fatty acyl-CoA esters (C12-C18), with the highest activity for palmitoyl-CoA. The apparent Km for palmitoyl-CoA was 47 microM for the microsomal enzyme and 17 microM for the mitochondrial-matrix enzyme. 2. The palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase activities of microsomal fraction had similar pH optima and were stimulated by dithiothreitol, but were affected differently by the addition of Pb2+, Mg2+, Ca2+, Mn2+ and cysteine. The two enzymes had different temperature-sensitivities. 3. The data strongly suggest that palmitoyl-CoA hydrolase and palmitoyl-L-carnitine hydrolase are separate microsomal enzymes, and that the hydrolysis of palmitoyl-CoA in the microsomal fraction and mitochondria matrix was catalysed by two different enzymes.     

ENZYMATIC ACYLATION OF 1-ALKYL-, 1-ALKENYL-AND 1-ACYL GLYCERO-3-PHOSPHORYLETHANOLAMINE IN DEVELOPING RAT BRAIN

Abstract— Rat brain particulate fractions were shown to acylate [³²P]1-alkyl- sn -glycero-3-phosphorylethanolamine (GPE). While the main product is 1-alkyl-2-acyl GPE, about 12 per cent of the radioactivity was also found in 1-alkenyl-2-acyl GPE. The acyl transferase activity was completely dependent on added ATP and CoA and it was localized mainly in the microsomal fraction. A comparative study of acyl transferase activities to 1-alkyl-, 1-alkenyl-, and 1-acyl GPE by crude mitochondrial fraction and microsomes of 10, 16 and 22-day-old rat brains showed a progressive increase in activity with development. In the 22-day-old rat brain the order of activity towards the three substrates is as follows: 1-acyl GPE ± 1-alkenyl GPE ± 1-alkyl GPE with a crude mitochondrial fraction and 1-acyl GPE ± 1-alkyl GPE ± 1-alkenyl GPE with microsomes.     

Sepharose-stearate as substrate for rat liver long-chain fatty acyl-CoA synthetase

Stearic acid coupled covalently to Sepharose 6B serves as substrate for thioesterification catalyzed by rat liver long-chain fatty acyl-CoA synthetase (ATP-forming) (EC 6.2.1.3). Availability as substrate is dependent upon the conservation of the free omega-terminal in addition to that of the free carboxyl function. The enzymatic overall formation of matrix-acyl-CoA in the presence of ATP and CoA as cosubstrates conforms to the stoichiometry reported for thioesterification of the free long-chain fatty acyl substrate. The preformed matrix-acyl-CoA serves as substrate for the backward synthetase reaction in the presence of AMP and PPi. The apparent Km values for ATP and CoA in the presence of the acyl matrix are similar to the respective Km values observed in the presence of the free acid substrate. The apparent Km for the acyl matrix is 10-fold higher (0.5 mM) than the apparent Km value for the free acid. The feasibility of enzymatic thioesterification of bound long-chain fatty acids implies that the exact nature of the bulky chain situated between the carboxy and omega-terminal plays a secondary role in defining the fatty acyl substrate specificity for long-chain fatty acyl-CoA synthetase. Also, dissociation of bound long-chain fatty acids does not constitute an obligatory preliminary step to fatty acid thioesterification.     

Synthesis of mixed diesters of ethanediol with N-acyl amino acids and fatty acids

Chemical synthesis of mixed diesters of ethanediol with N-acyl amino acids and fatty acids is described. The synthesis is performed in three steps: (1) preparation of N-acyl amino acids using fatty acid ester of N-hydroxyphthalimide as an acylating agent; (2) partial esterification of ethanediol with N-acyl amino acid, in tetrahydrofuran in presence of thionyl chloride; (3) further esterification of the monoester of ethanediol with a fatty acid, to a mixed diester, in presence of the same reagent.     

Xenobiotic acyl-CoA formation: evidence of kinetically distinct hepatic microsomal long-chain fatty acid and nafenopin-CoA ligases

Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin (K_i 53 μM) and ciprofibrate (K_i 1000 μM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform (K_m 6 ± 2.5 μM, (V_max 0.33 ± 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean K_i 1.7 μM, n = 5) and R-ibuprofen (mean K_i 10.8 μM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (V_max/K_m 0.057 ± 0.011 nmol/mg/min ± M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the K_m and K_i for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current ‘long-chain like’ xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.     

Species differences for stereoselective hydrolysis of propranolol prodrugs in plasma and liver

Species differences and substrate specificities for the stereoselective hydrolysis of fifteen O-acyl propranolol (PL) prodrugs were investigated in pH 7.4 Tris-HCl buffer and rat and dog plasma and liver subfractions. The (R)-isomers were preferentially converted to propranolol (PL) in both rat and dog plasma with the exception of isovaleryl-PL in rat plasma, although the hydrolytic activities of prodrugs in rat plasma were 5–119-fold greater than those in dog plasma. The prodrugs with promoieties (C(=O)CH(R)CH₃) based on propionic acid showed marked preference for hydrolysis of the (R)-enantiomers in plasma from both species (R/S ratio 2.5–18.2). On the other hand, the hepatic hydrolytic activities of prodrugs were greater in dog than rat, especially in cytosolic fractions. The hydrolytic activity was predominantly located in microsomes of the liver in rat, while the cytosol also contributed to hepatic hydrolysis in dog. Hepatic microsomal hydrolysis in dog showed a preference for the (R)-isomers except acetyl- and propionyl-PL. Interestingly, in rat liver all types of prodrugs with substituents of small carbon number showed (S)-preference for hydrolysis. The hydrolyses of (R)- and (S)-isomers of straight chain acyl esters in rat liver microsomes were linearly and parabolically related with the carbon number of substituents, respectively, while these relationships were linear for both isomers in dogs. Chirality 9:661–666, 1997. © 1997 Wiley-Liss, Inc.     

Catabolism of N-Acylethanolamine Phospholipids by Dog Brain Preparations

Abstract: N -Acylphosphatidylethanolamine, incubated with dog brain homogenate or microsomes, was hydroyzed to phosphatidic acid and N -acylethanolamine by a phosphodiesterase of the phospholipase D type. In the absence of F⁻, phosphatidic acid was further hydrolyzed to diacylglycerol and P_i while N -acylethanolamine was hydrolyzed by an amidase to fatty acid and ethanolamine. The phosphodiesterase showed an alkaline pH optimum and was also active towards N -acetylphosphatidyletha-nolamine, N -acyl-lysophosphatidylethanolamine, and glycerophospho( N -acyl)ethanolamine but showed little activity toward phosphatidylethanolamine and phosphati-dylcholine. Ca²⁺ stimulated slightly at low concentrations but inhibited at higher concentrations. Triton X-100 stim ulated the hydrolysis of N -acylphosphatidylethanol-amine, inhibited that of N -acyl-lysophosphatidyletha-nolamine and glycerophospho( N -acyl)ethanolamine, and had no effect on phosphatidylethanolamine or phospha-tidylcholine hydrolysis. The N -acylethanolamine hydrolase (amidase) was also present in the microsomal fraction and exhibited a pH optimum of 10.0. In addition to hydrolysis by the phosphodiesterase, N -acylphosphati-dylethanolamine was also catabolized by microsomal phospholipases A₁ and/or A₂ to N -acyl-lysophosphati-dylethanolamine, some of which was further hydrolyzed to glycerophospho( N -acyl)ethanolamine.     

Fatty acyl coupled catalase

Bovine liver catalase (hydrogen-peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was derivatized by 9″(10″)-[4′-{2-(4,6-dichloro-1,3,5-triazinyl)oxy}butoxy]stearic acid and the fatty acyl-coated enzyme was separated from native catalase and excess reagent by hydroxyapatite chromatography. The derivatization of catalase resulted in coupling the long-chain fatty acyl residues to lysine, histidine and arginine, while other amino acids remained essentially unaffected. The fatty acyl-coated enzyme was water soluble at pH > 7.0 but became octanol and ether soluble at pH < 6.5. The derivatized enzyme retained 50–80% of the catalatic- and peroxidative-specific activities. The free carboxyl function of the coupled long-chain fattyl acyl residues could serve as substrate for ATP-dependent CoA-thioesterification catalyzed by the rat liver microsomal long-chain fatty acyl-CoA synthase.     

Fatty acyl coupled catalase

Bovine liver catalase (hydrogen-peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6) was derivatized by 9"(10")-[4'-(2-(4,6-dichloro-1,3,5-triazinyl) oxy)butoxy] stearic acid and the fatty acyl-coated enzyme was separated from native catalase and excess reagent by hydroxyapatite chromatography. The derivatization of catalase resulted in coupling the long-chain fatty acyl residues to lysine, histidine and arginine, while other amino acids remained essentially unaffected. The fatty acyl-coated enzyme was water soluble at pH greater than 7.0 but became octanol and ether soluble at pH less than 6.5. The derivatized enzyme retained 50-80% of the catalatic- and peroxidative-specific activities. The free carboxyl function of the coupled long-chain fattyl acyl residues could serve as substrate for ATP-dependent CoA-thioesterification catalyzed by the rat liver microsomal long-chain fatty acyl-CoA synthase.     

Hydrolysis of phospholipids by a lysosomal enzyme

The phospholipid-hydrolyzing activity of rat liver lysosomes has been studied. These lysosomes contain a phospholipase that cleaves both fatty acid ester linkages of lecithin and of phosphatidyl ethanolamine and releases free fatty acids from both positional isomers of lysolecithin. The enzyme does not require calcium for maximum activity, and is inhibited by diethyl ether and sodium deoxycholate. Mercuric ions and cetyltrimethyl ammonium bromide also inhibit the hydrolysis. Compared with lipase activity, this enzyme is relatively stable to heat. The specific activity of the hydrolysis of lecithin by the lysosomal enzyme is considerably higher than those reported for mitochondrial and microsomal phospholipases. The enzyme resembles other hydrolases of the lysosome in that it has an acid pH optimum (pH 4.5). This enzymic activity is present in both the lysosomal soluble enzyme fraction and in the lysosomal membrane fraction. The enzyme may participate in the intracellular digestion of mitochondria that is carried out by the intact lysosome in vivo. Localized inflammation and changes in vascular permeability following tissue damage could be catalyzed by this phospholipase.     

Mass spectrometric analysis of long-chain esters of diols

Homologous series of synthetic long-chain monoesters and diesters of 1,2-ethanediol were analyzed by mass spectrometry, and the patterns of fragmentation were studied. Under electron impact saturated ethanediol monoesters yielded prominent ions characteristic of the short-chain diol, such as the rearranged ion formed by 2,3-cleavage (m/e 104) and the ion caused by 3,4-cleavage (m/e 117). Fragments characteristic of the constituent long-chain moieties were the acylium ions [RCO](+), [RCO - 1](+), and the ions [RC(OH)(2)](+). The mass spectra of ethanediol diesters exhibited very intense peaks due to the ions formed by loss of the acyloxy group, [M - RCOO](+), or one carboxylic acid, [M - RCOOH](+). These ions are distinctive for diol diesters. Precise mass measurements by high resolution mass spectrometry verified the composition of the ion fragments. Spectral studies of some monoesters and diesters of 1,3-propanediol, 1,4-butanediol, 2,3-butanediol, and also of some monounsaturated homologues, demonstrated that mass spectrometry is very suitable for the identification, distinction, and analysis of diol lipids.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Characterization of mono-, di- and triacylglycerol lipase activities in the isolated rat heart

The lipolytic activities of heart tissue towards full and partial acylglycerols were characterized. Tissue lysosomal, acid lipase activity (pH 4.8) was inhibited by high salt, protamine sulfate, NaF, MgATP, Triton X-100, serum and the esterase-inhibitor diethylparanitrophenyl phosphate. The tissue neutral triacylglycerol lipase activity (pH 7.4) was recovered predominantly in the microsomal and soluble fractions and exhibited essentially identical properties towards activators (serum, apolipoprotein C-II) and reagents (NaCl, Triton X-100, NaF, MgATP and diethylparanitrophenyl phosphate) relative to vascular lipoprotein lipase, except for protamine sulfate which increased the serum-stimulated neutral triacylglycerol lipase activity. Triacylglycerol hydrolysis at acid pH was incomplete, whereas at neutral pH full hydrolysis occurred. Myocardial mono- and diacylglycerol lipase activities, with pH optima of 8.0 and 7.4, respectively, were recovered in the microsomal fraction. They differed immunologically from neutral lipase and lipoprotein lipase and did not bind to heparin-Sepharose 4B. They were kinetically different, partially inhibited by NaCl and differentially affected by protamine sulfate. NaF, Triton X-100 and diethylparanitrophenyl phosphate. Our data suggest that endogenous hydrolytic activity against full and partial acylglycerols is mediated by separate enzymes.     

Formation of leukotriene B4-coenzyme A ester by rat liver microsomes

When leukotriene B4 (LTB4) was incubated with rat liver microsomal fraction in the presence of coenzyme A (CoA) and ATP, a more polar product (compound I) was detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product was identified as LTB4-CoA ester on the basis of ultraviolet spectrometry, alkaline hydrolysis followed by RP-HPLC, and fast atom bombardment mass spectrometry (FAB-MS). The activity forming LTB4-CoA ester was localized in the microsomal fraction. The reaction was proportional to the concentration of the microsomal protein with an optimal pH of 7.5-8.0 and completely dependent on CoA and ATP. Palmitic acid and myristic acid significantly inhibited the formation.