Biological Esterification of Steroids*

I. Introduction A. Obfuscating chemistry and the presumptive steroid acetates B. Sterol esters: substrates for the cholesterol side-chain cleavage enzyme II. Lipoidal Derivatives of Δ5-3β-Hydroxysteroids in the Adrenal and Ovary A. Endogenous steroid esters B. Biosynthetic esterification ofΔ 5-3β-hydroxy-steroids III. Lipoidal Derivatives of Steroids in Peripheral Tissues A. Esterification of steroid hormones B. Esterification of Δ5-3β-hydroxysteroids C. Endogenous esters of sex steroids D. Androsterone esters in breast cyst fluid IV. Lipoidal Derivatives Synthesized by Lecithin:Cholesterol Acyltransferase A. Blood B. Ovarian follicular fluid V. Steroid Esterases VI. Lipoidal Derivatives of Steroid Hormones in Insects VII. Biological Effects of Steroid Esterification A. Potent sex steroids B. Unique actions of the steroid esters in blood VIII. Conclusion FOR MORE than 150 yr, it has been known that sterols such as cholesterol are esterified with fatty acids in the body (reviewed in Ref. 1). The unique properties that make synthetic alkyl esters of steroids useful as pharmacological agents have been widely known for more than half a century (2–5). Nevertheless, the existence of naturally occurring fatty acid esters of steroids has been recognized only relatively recently. Many possibilities exist as to why fatty acid esters of steroids were not found at the same time that other endogenous steroid metabolites were identified. For example, the extracts from steroidogenic organs were usually saponified (6, 7) or partitioned between solvents to remove nonpolar lipids (8, 9), procedures that also would have destroyed or removed the steroid esters. In addition, steroid esters are not excreted in urine (10), the most accessible human source of steroid metabolites. As radiolabeled steroids became available for metabolic studies, nonpolar metabolites were frequently overlooked because nonpolar impurities are often present in the radioactive tracer or nonenzymatic hydrophobic artifacts may be formed during the incubation. Biosynthetic studies demonstrated that steroid esters, specifically acetates, can be produced artifactually during the isolation process. The 3-acetate of 5α-androstane-3β,17β-diol was identified in canine prostate and epididymis after arterial infusion of[ 3H]testosterone but it was not formed enzymatically. The steroid acetate was synthesized by transesterification in the solvent, ethyl acetate, which was used for extraction of the tissue (11). Likewise, dehydroepiandrosterone and 5-androstene-3β,17β-diol (Δ5-androstenediol) acetates were isolated and definitively identified by gas chromatography/mass spectroscopy (GC/MS) after the incubation of large concentrations of dehydroepiandrosterone (up to 100 μm) with mouse liver homogenates (12). The acetylation appeared to be enzymatic since it did not occur in heat-denatured tissues. However, the reaction was dependent upon extraction of the incubation medium with ethyl acetate. Apparently, an activated intermediate was formed that underwent transesterification with the solvent. Subsequently, it has been shown that biosynthetic acetylation can also result from esterase-catalyzed transesterification of organic acetates added to the incubation (13). Other nonenzymatic reactions that produce nonpolar products have also complicated the detection of biosynthetic esters. When 18-hydroxycorticosterone with a nonpolar formed that has properties of an However, it was found to be a of the detection of endogenous and biosynthetic steroid esters was often by or more of Nevertheless, as be were that nonpolar steroid esters but were overlooked or of the of the esterification of steroids was in it was found that a nonpolar was formed during of[ with of the or a mouse radioactive was by and it was identified as acetate by in as as by with added acetate. The that the isolation have been for nonenzymatic of the was the to have been but more have shown that esterified with fatty not acetate. The a result of the which are not as as and not other alkyl esters. alkyl esters often Other studies that acetates of at are formed during the incubation of[ with homogenates of the of the The for enzymatic esterification was The enzyme was an that for since such as were esterified at at the of the biosynthetic esters as acetates was studies in and other of the and have definitively identified the nonpolar metabolites as fatty acid esters and not The in the esters The of endogenous steroid fatty acid esters was of that were with of steroid esters, the In an and that of the steroidogenic used steroid as a to dehydroepiandrosterone the were shown to be and dehydroepiandrosterone as as it was found that to the and steroidogenic the cholesterol side-chain cleavage The cholesterol by extracts and as as in a naturally occurring which formed by the of cholesterol in a of the existence of the of the from to dehydroepiandrosterone was the enzymatic of cholesterol a was since of the or the of cholesterol to as substrates for the cholesterol side-chain cleavage enzyme it that the of a and would result in an for the of steroidogenic for a from the to the dehydroepiandrosterone to the of the cholesterol side-chain cleavage enzyme to the that with properties to fatty acid esters of steroids as substrates for steroidogenic substrates of of cholesterol were synthesized and with an enzyme to the that cholesterol to as a steroidogenic only did not cholesterol and cholesterol acetate, as The been synthesized and the that it would as a because it was widely that alkyl esters of cholesterol are not substrates for the cholesterol side-chain cleavage enzyme and that cholesterol esters in steroidogenic be to cholesterol can in the steroidogenic of the in the of by hormones the of the cholesterol by of cholesterol it was that the acetate of cholesterol as an for cholesterol side-chain cleavage acetate at the same at which cholesterol In of other cholesterol esters of were synthesized and as steroidogenic As the of the the of cleavage to cholesterol at which cleavage was 1). However, cholesterol as an which that it was of in with the cholesterol side-chain cleavage the that fatty acid esters of cholesterol as in it was that at of the fatty acid esters are not steroidogenic the the of steroidogenic a large of cholesterol fatty acid esters of which as substrates for the cleavage of cholesterol esters has been with a cholesterol side-chain cleavage the of a enzyme for esters cleavage of cholesterol esters by the cholesterol side-chain cleavage enzyme from The of in the as in cholesterol acetate, The of cholesterol to the of which of the of the esters are The from in Ref. cleavage of cholesterol esters by the cholesterol side-chain cleavage enzyme from The of in the as in cholesterol acetate, The of cholesterol to the of which of the of the esters are The from in Ref. The enzymatic of esters by the cholesterol side-chain cleavage enzyme the that cholesterol fatty acid esters in a The of the substrates and the fatty acid esters of were a was to from the a steroidogenic with the of an alkyl nonpolar from an organic of was produced and shown by with radioactive steroids to of nonpolar was with or that esters. The was for by acetylation with to the radioactive acetate, by was the that present would be found in only of were was as of the nonpolar of as and was more nonpolar than the did not produce of the nonpolar the of a of the present in the it appeared that a been was that the nonpolar was a fatty acid but the was the was the of The used to the of nonpolar steroid esters. it was that a unique steroidogenic been other were to for nonpolar metabolites of products formed in the of and dehydroepiandrosterone Lipoidal of of steroids were and the of was In the of to steroid was as the of a nonpolar to dehydroepiandrosterone are present in the the of the are present in the same of as the that of the Δ5-3β-hydroxysteroids are present in the and to and the ofΔ the are present in than the steroids in most in exist in than of the and fatty acid in the of The the and the the from B. with from The of of the and fatty acid in the of The the and the the from B. with from The of Lipoidal of steroids have also been found in the of a nonpolar from from and by of was by for the of steroids by were in the of the a of fatty acid esters of the The endogenous esters of steroids were and esters for of the it was shown that homogenates from the esterification of to but the esters were not to esters When was with the it was to which was esterified to the was that the of the and steroids found in the and were formed in a biosynthetic in which hydrophobic of were the was to with a of However, source of nonpolar metabolites was also in which steroid a the and were with dehydroepiandrosterone and was found that were nonpolar metabolites that are the steroid of nonpolar metabolites was that and were metabolites. Subsequently, produced by incubation of with was by and by spectroscopy The that a of fatty acid esters. esters were and the esters, and were present in the and of the The fatty acid of from that for esters of the other lipids in that be in the esterification of the source of in the was was that the fatty acid esters of that were identified have been synthesized by the cholesterol side-chain cleavage enzyme since esters of cholesterol are not substrates for the However, the esters that were identified been synthesized from and it was that other of the was to the endogenous by as an by incubation of[ with The biosynthetic identified as fatty acid esters of was added to an of endogenous The was a of The from the was to the was to for endogenous by by and of acetate after acetylation only with the the other were only of the was formed by esterification of was that the to of it that the in the other than the biosynthetic which of fatty acid esters. The in other have not been identified. to that were formed cleavage of the metabolites of Nevertheless, such a The existence of other has been For example, it was shown that fatty acid esters of at only substrates for the isolated from the incubation of[ with was with in the to products of a to of and were was that it that fatty acid esters esters it that steroidogenic fatty esters of steroids as substrates not In the of the endogenous of the steroids in the it was shown that steroids be esterified with fatty acids nonpolar was isolated from a incubation of the from with of The in was found to with of fatty acid esters and to as as fatty acids upon the of with esters. the in esterification of in the that the enzyme was an and that it was present in in the the incubation esterified but not that was in the enzymatic esterification of the However, were not The of esterification and the of of the that metabolites of other steroids also be was with of the the of was in of the tissue extracts The was found to have properties that are with esters at it was to to be the of a and than the was from but a the of esterification to be and not the was also by the that which not have a at was not a biosynthetic a relatively large of was as a and with of The was and an nonpolar that and of was by also most of the the to by and was of the was by which a of fatty acid esters. The also that the at and not at the The of the isolated in shown in of was and by which identified fatty acid esters of The fatty acids were and from the cholesterol esters and the present in the same tissue that be in the esterification of the of isolated from the incubation of with fatty acid esters of were of which was the most for of the the and esters have the same and the of the of the of esters are be as esters the of the The and esters were also by with from of isolated from the incubation of with fatty acid esters of were of which was the most for of the the and esters have the same and the of the of the of esters are be as esters the of the The and esters were also by with from the that be synthesized in breast and as a source of that formed in in and When acid or acid was in the the radiolabeled acids were was in a of that synthesized in breast of and The of the biosynthetic was the same of the steroid the esterification of in human breast and the of at concentrations of found a in the of the was removed from the In studies the was in breast and shown to be an present in the was that esterification of only at and esterification of that were esterified by the same However, the not have been since the of steroid was of in the of In other studies the esterification of as as of other was in the human breast The of the of was have studies with have not the of the and found that a for the from and an of the esterification of with a of and a of that the esterification not steroid However, not that substrates are esterified by the same since which not also a in Likewise, it been that the enzyme that as as the Δ5-3β-hydroxysteroids was In it a enzyme liver as a enzymatic found that the esterification of cholesterol and dehydroepiandrosterone as by to esters with fatty acid from the cholesterol esters. the the esterification of cholesterol but the esterification of the other studies demonstrated that from the that the other Many have been shown to have found that synthesized in of the and the from the of with The enzyme was shown to be an with a of for found that a of fatty acid as and that widely with the of and with the of the of the fatty acids not the of the esters in that have been found in in that the of the the most to which used to the of the esters in liver and that the enzyme an and that the synthesized was of esters: and The of the esters was in the the in the of fatty acids present in the tissues. a in with that as a of As the were the of the of metabolic to that as the from enzymatic of to the of a of can be the In of with a tissue that been for found that radioactive nonpolar metabolites were properties of that was a of fatty acid esters, at and the other at was more formed of the esters of were by and identified by The of the fatty acids and was found to be and to be the same as synthesized in the same that the esterification of in as as was the esterification at the was not was a The of fatty acid esterification the of of biosynthetic esters as and was isolated from the incubation of with The the of and which were added to the of the incubation medium as with from of biosynthetic esters as and was isolated from the incubation of with The the of and which were added to the of the incubation medium as with from the of[ by the and tissue of that of the appeared to be a nonpolar In a the properties of the nonpolar and found that it was to which that it was esterified at The was formed in or with the and it a between that of acetate and in the and found that it of fatty acid esters of the that been was isolated which of the of was also with of other esters. that the which to that the esters of a more were by of the fatty acid to a more during the isolation process. would also why it was that the were esterified with acetate also demonstrated that with a intermediate in nonpolar with was identified as a of and the other to be an of The biosynthetic of a was because the esterification of other steroids to a of fatty acid esters. the of and the esters was in and of was found to widely in tissues. was that esterified to and to other esters in However, in the same was a of at a to the esters. The of only of the In medium esters of were were in the The esterified of was also it was the The in the of the esters of and was in with the enzymatic that than the that the esters. of and esters synthesized in The steroid were isolated from in of and with tissue of the was at esters, only esters were of which with from and B. of and esters synthesized in The steroid were isolated from in of and with tissue of the was at esters, only esters were of which with from and B. it be to be the of has been the of to The that esterification be a by which be and in in breast to the of the of in breast ofΔ and dehydroepiandrosterone with human breast and to the of of steroids of nonpolar metabolites were of which upon products the esters were and The more was that the was esterified and that the was The of the to that it was a ofΔ the at and the was not and esters were also formed was with human breast In the with the of a was which was be the of from the the of the esters and were present in the studies that the of the of esters, in breast and that the fatty acid of the esters produced in the also Other studies of the esterification of in demonstrated that the incubation medium was most of the radioactive steroid in the was esterified incubation in the of added steroid to a of steroid that was produced by of the esters the for a that the and of were but found that the was esterified at the of the with the in which the in studies the of the ofΔ However, the was not by in of steroids and in to the are in the was not esterified it was and which were was In addition, it was by the and which were also that a of were esterified in it that not a for the studies also that the of at was not of a with the of the of were also esterification was not the of it was that the of of the steroids were the metabolites present in the that the a and and the esterification of and dehydroepiandrosterone in As most of the was present in the The biosynthetic was isolated and identified as fatty acid esters of after The fatty acid of was with that for the esters synthesized in in were in the of the esters of the did not to or to The synthesized in the was also with the lipids in the the fatty acid was other and were also shown to be a for the but not for the enzyme in breast was esterified at the of the steroids and were not The of were found in the to As the the The of only of the enzyme in the the in other also with was not and the in and liver of and and dehydroepiandrosterone as tissue in but not in the of the with steroids as In the the the dehydroepiandrosterone was the as the the liver the by the that are in the or for the Δ5-3β-hydroxysteroids not a to to in as an for of The was in human tissues. The steroid was in in and half that in was in and in The was also present in blood in blood and follicular fluid and in breast cyst it of the the of in and blood than that of that it be a of the of in after the of large of with a for after the the was and the metabolites from the were of the in the was present in the nonpolar by that the nonpolar was a between the and the of the most of the would have been in the of The that the of steroids in be used as a for the detection of a time after by the that esters would be the existence of a of as a endogenous by after and of the nonpolar found of in and of and in the of was in of the other that were blood and The from the was as by at an present in concentrations in with the be for the of the of of endogenous of steroid esters. When the of and from the of after was most of the from the in and be by In after the of in only after the was the of of was with that of the of of the endogenous esters of was more than that for esters and it the existence of a that the of the The of the of esters in the time after the were the was and the was and by for In the it that the has during that time was in the The are with from of The of the of esters in the time after the were the was and the was and by for In the it that the has during that time was in the The are with from of that was not found in because the same tissue in which esterification with fatty acids was also of because esters of have been found in tissue of the of the steroid the for in was more than that for in the was also found in it has been shown to be synthesized in a of of not the sex steroid esters can be to as the enzyme to be are that for tissue of enzymatic and of to possibilities at the esters are such substrates for and the to be the However, the hydrophobic may in the to of The that be of the In the enzyme in have a than the enzyme in other and would be of the esterification of of are known in steroid For example, are at of the and and the are in the of and Nevertheless, the that in the of and not that in human breast homogenates to a of a nonpolar and to for in human breast cyst fluid since it known to a of steroids and metabolites found of the of to of the in the cyst The was in it with the same time as an of an fatty acid The were by which the of esters as as The of was from by the of of esters were of and for more than of the The source of the esters was not it be that are synthesized in the breast cyst fluid by since such as are not esterified by enzyme In studies, and that steroids were with are nonpolar metabolites. that were the same metabolites as the that been found in the were with The of esterification dehydroepiandrosterone and and dehydroepiandrosterone were not demonstrated that of the are present in human the the of found in the in and and that ofΔ the concentrations of endogenous esters are to of was found that blood was in with and in and found that dehydroepiandrosterone was a in human and that most of the was to the As the esterification was by since it was by the acid has been shown that dehydroepiandrosterone and are esterified by appeared that the of steroids in blood were synthesized by Lipoidal did not to be been by that were synthesized by in were found only and not in the incubation However, studies of the that the esters may also be by the found that after with was a of esters in the with a in the esters in recognized that the of the esters in blood after have been the result of in the blood to of from the of the esters a However, in and that were esters, that the esters in blood are not products The formed in were present in the and When the the esterified and were a in it was that dehydroepiandrosterone esterified in human of the in the and in the of When dehydroepiandrosterone to by dehydroepiandrosterone and in blood in such a that the as would be the in blood was formed in by When was with the the blood of dehydroepiandrosterone and However, the esterification of but not as in blood in during dehydroepiandrosterone esterification with the of that may tissue of from also found that the of in blood with The in was than the in dehydroepiandrosterone in that with the esterification of dehydroepiandrosterone In studies of the of steroids with human of the steroids appeared to in a was that be an of esterification by synthesized by in the of human was isolated and by for steroid esters, it the of dehydroepiandrosterone studies that of the steroids of and appeared to have an of the were and were The of esterification of the steroids was to that The products of the in and were and by gas large of fatty acids were identified in the saponified the were to be of other of the that the and are fatty acid esters. As would be for products of the of fatty acids was the same as that of cholesterol esters isolated from the same the endogenous and present in were isolated from the and by The definitively that the in blood as fatty acid esters. the of and in the of blood that steroids are esterified by in the in and as as the that the steroids were to the other the same that cholesterol esters. The between by the cholesterol However, that biosynthetic esters of radioactive or dehydroepiandrosterone with an found that and were to of was the of was for the of cholesterol esters from to an to the of cholesterol esters to other but it did not the of and the of the steroid esters from that of cholesterol esters and not the of a in human nonpolar was isolated from and the after was by not be because of the that was by the of the and did not in recognized that did not have the to did that a for which would be for in Subsequently, the and that in in than in and in The of in blood than of in and in the has been in the blood of human but it that other have in the that was used in have not been to in human blood after a of not in the blood of and that produce a in also did not in blood it that the in same of the The between the esters, and with the of the which steroids in but not esterified by enzyme of acetates, acetate, have been to in human blood the of the esters as fatty acid esters, of esterification and the of steroid acetates as in the to be the of an for and that follicular fluid from with by after The endogenous was isolated and by not the of the esters was for was not because of the esters are to by However, that with to fatty acid esters. found that follicular fluid the of and that the enzymatic esterification was by the The biosynthetic was as a of fatty acid esters was to that for cholesterol esters in the steroid esters in in follicular fluid synthesized by that follicular fluid from human more than than the of in blood In more than in follicular of the in follicular fluid in the also the that which of the steroid from follicular the used to remove steroids from to steroid to that the of steroids be removed by and that may be present that be hormones also present in relatively large in human follicular fluid from with The of in the follicular was than in other tissue and a source for the isolation and of an endogenous of a of follicular fluid of were and the was by a of the was by that were identified by as acid esters. The of the esters, as by and after of the it was that the was from the than synthesized in by since a for and the of in the follicular fluid the same as the fatty acid was to that for the in follicular fluid that was synthesized by were to that and have been synthesized by Subsequently, the was between the fatty acid of endogenous in follicular and that of synthesized in by in the same follicular fluid or in the were the synthesized in isolated from the follicular fluid a in follicular synthesized by The relatively concentrations of in follicular fluid a result of the of the metabolic by the of the and the of the to of the The of in the follicular fluid