natural occurrence of free fattv aldehydes in bovine ... · natural occurrence of free fattv...

Natural occurrence of free fattv aldehydes in bovine cardiac muscle

John R. Gilbertson, Ronald C. Johnson, Rose A. Gelman, and Carol Buffenmyer

Department of Pharmacology and Physiology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213

Abstract Free fatty acids, aldehydes, alcohols, and 1-0-alkyl and alk-l-enyl glycerols were identified and quantified in lipid extracts from bovine cardiac muscle.

Although a number of components present in the free fatty aldehydes were also noted in the fatty chains in the l-O-alk-1- enyl glycerols, a direct qualitative similarity did not exist as would be expected if the free fatty aldehydes were artifactual in origin. Also, a qualitative similarity did not exist between the fatty chains of the 1-0-alkyl and alk-1-enyl glycerols. This lat- ter observation would suggest a mechanism other than biodehydrogenation of the alkyl ethers for the origin of the alk-1-enyl glycerols.

Free fatty aldehydes were distributed evenly between the 105,000 g supernatant and particulate fractions of cardiac muscle, while the 1-0-alk-1-enyl glycerols were associated primarily with the particulate fraction. Free fatty alcohols were noted only in the supernatant fraction, while the 1-0-alkyl glycerols were present in both fractions.

Supplementary key words 1-0-alkyl glycerois . alk-1-enyl glycerols . fatty aldehydes . fatty alcohols . thin-layer chromatography . gas-liquid chromatography . column chromatography

F R E E FATTY ALDEHYDES have previously been reported as trace lipids in mammalian tissues, sea animals, and plants (1-7). Although these fatty compounds have been recognized in widely divergent tissues, investiga- tions concerning their metabolic origin have been limited. Biochemical studies have indicated that enzyme systems capable of catalyzing the reduction of fatty acids to aldehydes are present in rat brain, bovine cardiac muscle, bacteria, and yeast (8-11). In the microorganism Euglena gracilis, fatty aldehydes were not

Abbreviations: TLC, thin-layer chromatography; GLC, gas- liquid chromatography.

observed as such but occurred as enzyme-bound intermediates in the reduction of fatty acids to alcohols

Although this evidence indicates that free fatty aldehydes originate by the enzymatic reduction of fatty acids, it is also known that fatty aldehydes arise from the catabolism of sphinganine or in the oxidation of 1-0- alkyl glycerols to fatty alcohols and glycerol (13-16). Evidence that enzymatic hydrolysis of the alk-1-enyl ether linkage of the phospholipids will result in the ap- pearance of fatty aldehydes has also been obtained (17,18).

Recently, it has been questioned whether free fatty aldehydes occur at all in mammalian brain tissue (19), and it was suggested that this lipid type arises from the autolysis or hydrolysis of the 1-0-alk-1-enyl glycerols

The studies mentioned previously indicating that free fatty aldehydes may arise by several mechanisms make it important to evaluate their origin in cells. This is of special significance since the fatty aldehydes may be metabolic intermediates in the biosynthesis of free fatty alcohols.

The importance of free fatty alcohols in the biosynthesis of the 1-0-alkyl glycerols has been defined (21-27). The involvement of the free fatty aldehydes is more obscure, although the incorporation of this lipid type into the 1-0-alkyl and 1-0-alk-1-enyl glycerols has been demonstrated in intact animals (14, 28, 29). That the fatty aldehyde may be first reduced to a fatty alcohol prior to utilization in glycerol ether biosynthesis has been proposed (1 4).

The information reported here concerns the quantitation of the free fatty acids, aldehydes, alcohols, and 1-0-alkyl and alk-1-enyl glycerols from the same lipid extract. Qualitative identification of several of these lipid types was also achieved.

(1 2).

(20, 21).

Journal of Lipid Research Volume 13, 1972 491

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EXPERIMENTAL PROCEDURES

Extraction of tissue lipids

Bovine cardiac muscle was obtained fresh from a local slaughterhouse and stored in ice during transit. The procedure of tissue preparation and lipid extraction has been described (2). Throughout this study all of the lipid residues were routinely blanketed with nitrogen and dissolved in nitrogen-equilibrated n-heptane except for the phospholipids, which were dissolved in nitrogen- equilibrated chloroform.

Resolution of lipid classes

The total lipid extract was divided into two equal aliquots; all lipid types but the free fatty aldehydes were isolated from one aliquot by column chromatography. Free fatty aldehydes were isolated from the second aliquot by thin-layer chromatography.

Column chromatography

Aliquots containing 200-250 mg of lipid were removed from the total lipid extract, concentrated under vacuum to approximately a 5-ml volume, and applied to a column containing 18 g of n-heptane-equilibrated silicic acid (30). Column development was initiated with 250 ml of 1% diethyl ether in n-heptane to elute cholesteryl esters, free fatty aldehydes, and other lipids (2, 30). Triacyl glycerols, 1-0-alkyl and alk-1-enyl diacyl glycerols, and free fatty acids were eluted in the next fraction with 4y0 diethyl ether in n-heptane. The column was then developed with 300 ml of 10% diethyl ether in n-heptane to elute free fatty alcohols, cholesterol, and any residual free fatty acids. Following this, diacyl and monoacyl glycerols, alkyl and alk-1-enyl monoacyl glycerols, and alkyl and alk-1-enyl glycerols were eluted with 300 ml of diethyl ether. Phospholipids were eluted with 400 ml of methanol (2,30).

Thin-layer chromatography

Free fatty aldehydes. The secocd aliquot from the total lipid extract was concentrated to a small volume, and the free fatty aldehydes were isolated on neutral thin layers of silica gel G prewashed with diethyl ether. In this instance, hexane-isobutanol-methanol 100 : 3 : 4 was used as the developing solvent (1, 2). After the chromatogram was developed, it was dried under nitrogen and sprayed with o.O5y0 rhodamine 6G in 80% methanol. The lipid migrating as the free fatty aldehyde standard was scraped from the plate and eluted with n-heptane. The n-heptane was immediately removed under vacuum in a rotary evaporator, leaving a lipid residue which was then diluted to a known volume. From this, aliquots were taken for quantitation and gas- liquid chromatography.

Free fatty alcohols. Any free fatty acids present in the column fraction eluted with 10% ether in n-heptane were removed by treating with resin as described in the section on fatty acids and were subsequently added to the acids previously eluted from the silicic acid column with 4% diethyl ether in n-heptane. The lipids that did not adsorb to the resin were concentrated to dryness under vacuum. The residue was dissolved in n-heptane, and the free fatty alcohols were separated from cholesterol and other lipid contaminants by TLC (5). Hydroxy waxes, esters of hydroxy fatty acids and fatty alcohols, and alkane diols were some of the possible lipid contaminants. Evidence that the free fatty alcohols or the 1-acetoxy alkane derivatives can be separated from these contaminants is presented below. The free fatty alcohols eluted from a silicic acid column were resolved from cholesterol and other lipid compounds by TLC; a chromatogram demonstrating the separations achieved is presented in Fig. 1. In this system the free fatty alcohols are resolved from cholesterol and the hydroxy fatty acid, ricinoleic acid, but still may be contaminated with glycol ethers and hydroxy waxes. These compounds were acetylated (31) and resolved via TLC. Fig. 2 demonstrates that the 1-acetoxy alkanes are readily separated from the acetates of these other compounds. While diglycerides were not expected to be eluted from a silicic acid column in the fraction containing the free fatty alcohols (30, 32), this lipid type does migrate during TLC with a mobility similar to a fatty alcohol. From the data presented in Fig. 2, it is apparent that the 1-acetoxy alkanes can be readily resolved from the acetylated 1,2- or 1,3-diacyl glycerols in this TLC system.

Recovery of the free fatty alcohols in the isolation procedure described here was assessed by adding various amounts of [1-14C]hexadecan~l to the total lipid extracts prepared from different bovine hearts and isolating the fatty alcohols as described. Under these conditions, 92% (range 90-95y0, n = 6) of the total radioactivity applied as the free fatty alcohol was recovered as the 1-acetoxy alkane.

Free fatty acids and 1-0-alkyl and alk-1-enyl glycerols derived from the neutral lipid fraction. Silicic acid column fractions containing the tri-, di-, and monoacyl glycerols, the 1-0-alkyl and alk-1-enyl glycerols, and free fatty acids were concentrated to dryness under vacuum, and the fatty acids were separated from the remaining neutral lipids by adsorption onto an ion exchange resin (33, 34). Free fatty acids were eluted from the resin with chloroform-methanol 1 : 1 that was 1 N with HC1. Sufficient chloroform and 0.1% NaCl were added to the eluate to achieve the conditions of a salty wash (35). The free fatty acids were recovered in the chloroform phase, methylated, and quantitated (31,36).

492 Journal of Lipid Research Volume 13, 1972


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Recovery of free fatty acids and triacyl glycerols in the ion exchange procedure described previously was assessed with standard mixtures of [l-14C]palmitic acid and tristearin containing 24.5-180 Mmoles of palmitic acid and 48.6-286.0 pmoles of tristearin. Recovery of the free fatty acids was based on a comparison of the radioactivity present in the untreated mixture and the lipid eluted from the ion exchange resin. Recovery of the triacyl glycerols was based upon a comparison of the ester groups present in the untreated mixture and the lipids not adsorbed to the resin. Under these conditions the average recovery of tristearin was 98.1 f 4.1y0 SD

(n = 13) and that of palmitic acid 101.0 f 4.2% SD

(n = 13). The neutral lipids not adsorbed to the resin were

saponified, and the nonsaponifiable lipids were extracted from the alkaline aqueous phase. The 1-0-alkyl and alk-1-enyl glycerols were separated from other non- sapotifiable lipids by TLC, and the total glycerol ether content, 1-0-alkyl and 1-0-alk-1-enyl, of the purified lipid was determined (37). Neutral glycerides were quantitated by assaying the glycerol content of the aqueous phase (38).

A separate aliquot was taken from the purified total glycerol ethers for acid hydrolysis (39). The 1-0-alkyl glycerols and the aldehydes derived from the alk-1-enyl glycerols were resolved by TLC (2). Aliquots were taken from these lipid types for GLC and quantitation.

Reproducibility of the procedures employed for the isolation of free fatty aldehydes, alcohols, and acids. The reproducibility of the procedures used for the isolation of the free fatty aldehydes, free fatty alcohols, and free fatty acids was assessed by isolating these lipid types from five random pieces, weighing 12.9 f 0.4 g (sD), of the same bovine ventricle. The five pieces of tissue yielded an average of 411 f 27 mg of total lipid containing 0.53 f 0.04 pmole of free fatty aldehydes, 67.23 f 3.39 pmoles of free fatty acid, and 0.55 f 0.05 pmole of free fatty alcohols. From these data and those presented previously it is apparent that the results obtained in the isolation of these lipid types are both quantitative and reproducible.

Alkyl acyl and alk-I-enyl acyl glycerophosfihatides. After elution of the phospholipids from the silicic acid column, solvent was removed under vacuum and the residue was dissolved in a known volume of chloroform. From this solution aliquots were taken to determine both the 1-0-alk-1-enyl glycerol content and the lipid phosphorus content (2). A separate aliquot of the phospholipids was concentrated to dryness and the residue was treated with LiAlH4 (37). The resulting 1-0-alkyl and alk-l- enyl glycerols were isolated by TLC and hydrolyzed in acid. The fatty aldehydes derived from the l-O-alk-1- enyl glycerols were resolved from the 1-0-alkyl glycerols

by TLC. Aliquots were taken from each lipid type for GLC and GLC quantitation.

Gas-liquid chromatography

The individual lipid classes were characterized by GLC. Free fatty aldehydes and the fatty aldehydes formed by acid hydrolysis of the 1-0-alk-1-enyl glycerols were chromatographed as such. Free fatty alcohols were analyzed as 1-acetoxy alkanes and the 1-0-alkyl glycerols as 2-acetoxy acetaldehydes (40).

The instrument used in these analyses, the methods of peak identification, and calculation of percentage composition have been described (2). In this instance standards were added to natural mixtures and co- chromatography of the components was also employed to establish the assigned designations. In these analyses 6-ft U-shaped silanized borosilicate glass columns (1/8-inch I.D.) containing 3y0 OV-101 on silanized 100-1 20 mesh Gas-Chrom Q or 1 5y0 DEGS on silanized 80-100 mesh Gas-Chrom P were used. Fatty aldehydes, 1-acetoxy alkanes, and 2-0-acetoxy acetaldehydes were chromatographed at 150, 170, and 180"C, respectively, with OV-101, and at 160, 180, and 180°C with DEGS as the liquid phase. The flash evaporator and detector temperatures were routinely maintained 20°C higher than the column oven temperatures.

Preparation and analysis of subcellular fractions

Bovine cardiac muscle free of extraneous tissues was sectioned into I-cm cubes and homogenized in sufi- cient 0.25 M sucrose in 0.05 Tris buffer, pH 7.8, to give a 25y0 homogenate. The tissue was initially homogenized at low speed in a Waring Blendor for 30 sec and then strained through cheesecloth to yield a total filtrate. This filtrate was centrifuged at 700 g for 20 min, and the supernatant fraction was divided into two equal parts. One part was centrifuged at 105,000 g for 60 min to yield a total particulate and supernatant fraction (41). Corresponding aliquots were removed from the 700 g supernatant solution, the 105,000 g particulate fraction, and the 105,000 g supernatant fraction, and the lipid was extracted as described above. After the removal of an aliquot for the determination of lipid weight, the remaining lipid extract was divided into equal parts and individual lipid types were isolated as before.

Aliquots were also taken from the above fractions for protein determination (42).

Materials

All solvents were reagent grade and, with the excep- tion of methanol, were redistilled before use. The solid support and liquid phases used for GLC, as well as the fatty aldehydes, fatty alcohols, and 1-0-alkyl glycerols used as chromatographic standards, were obtained from

Gilbertson, Johnson, Gelman, and Buffenmyer Aldehydogenic Lipids, Alcohols, and Ethers 493


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Applied Science Laboratories Inc., State College, Pa. The sources of the materials used for the various column and thin-layer chromatographic procedures have been described (2). Standard 2-octadecoxy ethanol was prepared by periodate oxidation of 1-0-octadecoxy glycerol and reduction of the purified octadecoxy acetaldehyde with LiAlH4 (39, 40). The resulting glycol ether was purified by TLC and the compound was verified by infrared spectroscopy. n-Butyl ricinoleate was prepared by heating ricinoleic acid at 60" for 2 hr in n-butanol containing 2% HC1 (w/w). The resulting ester was purified by TLC and characterized by infrared spectroscopy.

RESULTS

Quantitative distribution of the free fatty acids, free fatty aldehydes, free fatty alcohols, and 1-0-alkyl and alk-1-enyl glycerols from bovine cardiac muscle

Three different bovine hearts were analyzed for the lipid types under consideration, and the results of these analyses are in Table 1. From the data it is evident that the free fatty aldehydes and free fatty alcohols are present in trace amounts in cardiac muscle, the free fatty

alcohols being present in amounts two to four times greater than the free fatty aldehydes.

With respect to the total aldehydogenic lipids present, free fatty aldehydes and 1-0-alk-1-enyl glycerols, the majority are present as 1-0-alk-1-enyl glycerophosphatides. About 1% of the total aldehydogenic lipids are present as free fatty aldehydes. Although the free fatty aldehydes are a minor fraction of the total aldehydogenic lipids, in the neutral lipid fraction the free fatty aldehydes are present in amounts similar to the 1-0-alk-1-enyl glycerols.

Qualitative distribution of the free fatty aldehydes, free fatty alcohols, and 1-0-alkyl and alk-1-enyl glycerols from bovine cardiac muscle

The distribution of these several lipid types isolated from hearts 1 and I1 is presented in Table 2. From the data it is apparent that four fatty species, penta-, hexa-, hepta-, and octadecanal, account for 90% of the free fatty aldehydes, hexadecanal being the major component present. The free fatty alcohols differ from the free fatty aldehydes in that dodecanol and tetradecanol are present in increased amounts in the free fatty alcohols, and heptadecenol, which is a minor fatty alcohol, is a

TABLE 1. Free fatty acid, free fatty aldehyde, free fatty alcohol, and alkyl and alk-1-enyl glycerol ether content of bovine cardiac muscle

Neutral Lipida Phospholipid' - Tissue Free Fatty: Glycerol Ethers Glycerol Etners

Expt. Wet Wt Lipid 'Acids Aldehydes Alcohols Total Alk-l-enyl Glycerol Alkyl Alk-l-enyl Lipid P

I 15.0 2.4 10.8 0.07 0.12 0.39 0.13 51.4 0.70 7.90 56.3

111 43.9 2.7 10.6 0.05 0.12 0.47 0.15 42.4 0.63 5.58 78.1 I1 12.8 3.1 16.8 0.03 0.13

a The amount of each lipid type present is expressed as pmoles/100 mg total lipid.

TABLE 2. Percentage distribution of the free fatty aldehydes, alcohols, and 1-0-alkyl and alk-1-enyl glycerol ethers in bovine cardiac muscle

~~

Glycerol Ethers Alkyl Alk-1-Enyl

Free Fatty: Neutral Phospho- Neutral Phospho-

Shorthanda Aldehyde Alcohol Lipid lipid Lipid lipid Desienation I I1 I I1 I I1 I I1 I I1 I I1

12:o 14:O 15: 1 15:O 16: 1 16: 0 17: 1 17: 0 18: 1 18:O

@ 6 . 0

11.3 2 . 2

49.8 2 .0

10.7

18.0

2.6 11.4 2.8 23.3

3.7 1.1 11.2

62.2 42.6 3.6 9.8 1 .2

12.3 tr 11.5

16.5 14.7 1.6 tr tr

5 . 5 3 .3 2 .3 2 .8 8 . 0 3 .5 1 .1 2 .8 2 .2

3 .5 1 .5 2 .5 2 .3 27.0 44.8 33.8 41.5 46.9 39.8 27.4 42.0 47.9

6.3 3 .3 5 .1 5 .4 1 .4 4 .5 6 .3 3 .4 2.4

23.2 28.2 19.5 31.9 15.9 11.1 10.5 7 .0 7 .5 9.2 27.0 47.1 27.5 37.2 27.2 45.8 33.6 28.4

0 Number of carbon atoms: number of double bonds. 6 tr, less than 1 yo of the total.



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TABLE 3. Subcellular distribution of the free fatty aldehydes, fatty alcohols, and 1-0-alkyl and alk-1-enyl glycerophosphatides

Tissue Free Fatty Free Fatty Glycerophosphatides Expt. Wet Wt Protein Lipid Aldehydes Alcohols Alkyl Alk-1-enyl

g mg % pmolcs pmoles w a l e s pmoles A

Total filtrate 14.9 1526 2.4 0.41 0.30 1.95 20.6 700 g supernate 330 0.26 0.15 1.32 8.6 105,000 g

Particulate fraction 139 0.13 0.88 6.6 Supernatant fraction 202 0.11 0.17 0.36 1.12

B Total filtrate 16.7 1651 2.7 0 .68 0.80 2.03 28 . O 700 g supernate 458 0.32 0.35 1.34 12.0 105,000 g

Particulate fraction 184 0.16 0.94 10.4 Supernatant fraction 247 0.11 0.34 0.44 2.2

significant component of the free fatty aldehydes. The reason for the variation in the pentadecanol, hexadecanol, and octadecenol content of the two hearts is not understood.

The fatty chains of the 1-0-alkyl glycerols were accounted for by those components corresponding to 1-0-octadecoxy, hexadecoxy, and octadecenoxy glycerols, and thus differed to a large extent from the 1-0- alk-1-enyl glycerols, where pentadecanal, heptadecanal, and several monounsaturated species were present in readily detectable amounts.

Subcellular distribution of the free fatty aldehydes, fatty alcohols, and 1-0-alkyl and alk-1-enyl glycerophosphatides

A cell-free homogenate, 700 g supernate, was prepared from cardiac muscle and further separated into particulate and soluble fractions by centrifugation at 105,000 g. Lipid was extracted from each fraction and analyzed for free fatty aldehydes, fatty alcohols, and 1-0-alkyl and alk-1-enyl glycerophosphatides. The results of these analyses are presented in Table 3.

From these data it is apparent that the free fatty aldehydes are present in both the 105,000 g particulate and supernatant fractions obtained from the 700 g supernatant fraction. While the 1-0-alkyl and alk-l- enyl glycerophosphatides are present in both the 105,000 g particulate and 105,000 g supernatant fractions, the majority of the glycerol ethers are associated with the mitochondrial-microsomal fraction. The subcellular distribution of the free fatty aldehydes is not the same as that of the 1-0-alk-1-enyl glycerols, since approximately 80% of the total alk-1-enyl ethers in the 700 g supernatant fraction are localized in the 105,000 g particulate fraction while the free fatty aldehydes are quite evenly distributed in the two subcellular fractions.

The amounts of 1-0-alkyl and alk-1-enyl glycerols reported in Table 3 represent those observed following

LiAlH4 reduction of the mixed phospholipids and purification of the total glycerol ethers by TLC.

DISCUSSION

Methods previously utilized in this laboratory for the isolation of free fatty aldehydes and total glycerol ethers have been expanded to permit the isolation of free fatty acids and free fatty alcohols from the same lipid extract (1, 2, 38). In instances in which the methods employed had not previously been evaluated, they were shown to be quantitative and reproducible.

In this study, both the free fatty aldehydes and free fatty alcohols were isolated in trace amounts free from other contaminants. The free fatty aldehydes accounted for about 1% of the total aldehydogenic lipids of cardiac muscle but !joy0 of the aldehydogenic neutral lipids. Similar amounts of free fatty aldehydes have been noted before in bovine cardiac muscle (2).

The free fatty alcohols were detected in amounts greater than those of the free fatty aldehydes from the same tissue and in greater concentrations than those reported previously for bovine cardiac muscle (32, 43). At present this discrepancy cannot be explained except to note that within the same tissue individual lipid species have been fou,ld to vary as much as 2-10-fold when one subcellular compartment is compared with another (44, 45). If such a change can occur due to localization of one lipid type in a particular subcellular compartment, some variation should be expected when age, diet, and species are not considered. That the isolation procedure employed did not select specific fatty alcohols, i.e., saturated, or that spuriously high values were due to the presence of cholesteryl acetate or other lipid contaminants was ruled out by several observations. First, GLC of the fatty alcohols eluted from the silicic acid column gave the same qualitative distribution as their purified 1-acetoxy alkanes. Second,

Gilbertson, Johnson, Gelman, and Bufjenmyer Aldehydogenic Lipids, Alcohols, and Ethers 495


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when fatty alcohols obtained from LiAlHd reduction of total bovine phospholipids were mixed with cholesterol and then treated in the same manner as a free fatty alcohol and cholesterol fraction, GLC of the isolated alcohols again gave the same qualitative distribution as the 1-acetoxy alkane derivatives, and polyunsaturated moieties were detected. Third, the purity of the acetoxy alkanes was evaluated by analyzing these compounds by GLC under conditions previously utilized to identify cholesterol (46). Under these conditions, high molecular weight components such as cholesteryl acetate or the acetates of 2-hexadecoxy or 2-octadecoxy ethanol were not observed. Finally, evidence for the purity of the 1- acetoxy alkanes may be obtained by comparing the TLC mobilities of the free fatty alcohols and the derived acetoxy alkanes with those of other possible contaminants and their acetates, as seen in Figs. 1 and 2.

While the emphasis in this study was not placed on the free fatty acid content of cardiac muscle, it was noted that the free fatty acid content observed here is similar to that reported by others for bovine cardiac muscle (44). The 1-0-alk-1-enyl glycerol content of the phospholipids is less than that observed previously in bovine and human cardiac muscle (44, 3) but falls within the range reported for rat and mouse heart (47, 48). A considerable variation with respect to the 1-0-alk-1-enyl glycerol content of cardiac muscle in adult members of different species has been noted before (48). That age of the animal as well as species may account in part for the variation in the 1-0-alk-1-enyl glycerophosphatide content of the heart is indicated by the observation that the 1-0-alk-1-enyl glycerophosphoryl choline concen- tration of dog and cat cardiac muscle increased as the animal matured (49). As noted previously, l-O-alk-1- enyl glycerols are the major ether species present in the phospholipid fraction (37). In the neutral lipid fraction the glyceryl ethers are present in trace amounts (34,49). In contrast to the results obtained in rat cardiac muscle and in agreement with those observed before in calf heart, the 1-0-alkyl glycerols were the major glyceryl ether type observed in the neutral lipids (32,

Although the data are not reported here, qualitative analysis of the free fatty acids indicated the presence of polyunsaturated species such as linoleic and arachi- donic acids in major amounts as well as smaller amounts of longer-chain compounds. Similar data for the free fatty acids of bovine cardiac muscle have been presented (44).

In contrast, the free fatty aldehydes and fatty alcohols were characterized by an absence of polyunsaturated species. A similar qualitative difference has been noted before with respect to the free fatty acids and fatty alcohols isolated from malignant tissue and has been


37).

, -. ”

h C

FIG. 1. Thin-layer chromatogram of standards on silica gel G. Solvent system, diethyl ether-hexaneglacial acetic acid 70: 30: 1 ; indicator, iodine vapor. Components of each lane from the origin to the front are: A , hexadecanol; B, cholesterol; C, cholesterol plus octadecenol; D, cholesterol plus 2-octadecoxy ethanol; E, n- butyl ricinoleate plus ricinoleic acid.

0 H

. ”

t I

FIG. 2. Thin-layer chromatogram of standards on silica gel G. Solvent system, hexane-diethyl ether-acetic acid 90: 10: 1 ; indicator, iodine vapor. Components of each lane from the origin to the front are: F, I-acetoxy hexadecane; C, acetates of a 70:30 mixture of 1,2- and 1,340lein; H, 2-octadecoxy-1-0-acetoyl ethanol; I , n-butyl-12acetoyl ricinoleate.

taken as the basis for suggesting a substrate specificity for the enzymes involved in the biosynthesis of fatty alcohols (50). Substrate specificity has been observed before with horse liver alcohol dehydrogenase as well as with the retinal reductase isolated from rat intestinal mucosa (14,51).

The qualitative analysis of the free fatty aldehydes of bovine cardiac muscle reported here is similar to the results published previously in that hexadecanal and octadecanal are the major free fatty aldehydes detected (2). The results differ from those reported initially in that in this instance heptadecanal and tetradecanal are present in greater amounts.

Qualitative analysis of the free fatty alcohols of malignant tissue has indicated that hexadecanol, oc- tadecanol, and octadecenol accounted for almost all of the


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fatty alcohols present (51). Similar analysisof the free fatty alcohols in bovine and porcine heart and brain indicated not only the presence of fatty alcohols with 16 and 18 C atoms, but also the presence of heptadecanol, eicosanol, and docosanol in significant amounts (45). These species were not noted as components of the fatty alcohols from malignant tissue. In this study, long-chain fatty alcohols greater than 18 carbon atoms in chain length were not detected, but short-chain species with 12-16 carbon atoms were noted in considerable amounts. Previous analysis of the alcohol moiety of the wax esters isolated from the preputial glands of mice has indicated that tetradecenol, pentadecanol, and hexadecenol are present as major components (52).

At present, the reason for the variation in chain length of the free fatty aldehydes and alcohols in these several studies is not known. That part of the variation in composition may be due to the activity of more than one enzyme is suggested by the fact that free fatty aldehydes were detected in both the 105,000 g supernatant and particulate fractions while free fatty alcohols were observed in only the 105,OOOg supernatant fraction.

1-0-Octadecoxy, hexadecoxy, and octadecenoxy glycerols were observed to account for the majority of the 1-0-alkyl glycerols in both the neutral and phospholipid fractions in this study. This observation is in agreement with results obtained previously in human heart and other tissues (20,25,51).

The fatty chains of the 1-0-alk-1-enyl glycerols observed here were similar to those noted previously in bovine cardiac muscle except that in this instance branched-chain moieties were not detected in trace amounts and the presence of the odd-chain monoenes, pentadecenal and heptadecenal, were noted in greater than trace amounts (20).

Previously it has been indicated that the presence of free fatty aldehydes in lipid extracts may be artifactual due to the autolysis or chemical hydrolysis of the 1-0- alk-1-enyl glyceryl ether bond (20). This conclusion was based on an observed qualitative similarity between the fatty chains of the free fatty aldehydes and 1-0-alk-l- enyl glycerols isolated from human cardiac muscle. It is interesting to note that other workers analyzing the same lipid types from human cardiac muscle obtained different results and reached the opposite conclusion (3).

Free fatty aldehydes have also been suggested to arise from the enzymatic hydrolysis of the 1-0-alk-1-enyl glyceryl ether bond (21), an enzymatic activity which has previously been demonstrated in mammalian tissue (17, 18).

In a similar vein, fatty alcohols are known to be one of the possible end products resulting from the catabolism of the 1-0-alkyl glycerols (16). Yet a qualitative similarity

between the fatty chains of the free fatty alcohols and 1-0-alkyl glycerols served as the initial basis for suggesting a precursor-product relationship between these lipid types, a suggestion that was soon established and confirmed (21,24,52).

While free fatty aldehydes are suggested to arise from the chemical or enzymatic hydrolysis of the 1-0-alk-1- enyl glycerols, data to support this conclusion have not been published (20, 21). A qualitative similarity, as noted previously, does not necessarily preclude a catabolic relationship.

Recently, a study questioning the existence of free fatty aldehydes in brain tissue and implying their absence in other tissues has appeared (19). Other studies indicating that free fatty aldehydes are biosynthesized and metabolized in this organ are available (8,53).

In this study, observations that would reconfirm the natural occurrence of the free fatty aldehydes in cardiac muscle have been obtained. First, the qualitative dis- tributions of the fatty chains of the free fatty aldehydes and the 1-0-alk-1-enyl glycerols were not identical (Table 2). Although generally the same fatty chains are present in both lipid types, octadecenal and heptadecanal, both components of the alk-1-enyl glycerols are either not detected in the free fatty aldehydes or are present in different proportions, Tetradecanal is present primarily as a free fatty aldehyde. Second, the data reported in Table 3 indicate that the 1-0-alk-1-enyl glycerol content of the various subcellular fractions is 10-40 times greater than that of the free fatty aldehydes present in the same fraction, yet the free fatty aldehyde content of each fraction is quite similar. If hydrolysis were occurring, one would expect the free fatty aldehyde content to be increased in fractions containing large amounts of I-0-alk-1-enyl glycerols.

That the free fatty aldehydes noted here are not catabolic products derived from the 1-0-alkyl glycerols is apparent from the qualitative difference observed between the fatty chains of the free fatty aldehydes and the 1 -0-alkyl glycerols (Table 2).

Biosynthesis of the 1-0-alkyl glycerols has been noted previously in mammalian tissues both in vivo and in vitro (5, 13, 14, 19, 21-29). In vivo biosynthesis of the 1-0-alk-1-enyl ether bond has been shown to proceed via biodehydrogenation of the corresponding 1-0-alkyl glycerol (14,21,24,27,29).

Information suggesting that the alk-1-enyl ether bond may be synthesized by another mechanism has been recently published (19, 54, 55). The results here, and the observations of other workers (20) would also suggest that not all the 1-0-alk-1-enyl glycerols are formed by biodehydrogenation of the corresponding alkyl glycerol. Although the major fatty chains of the 1-0- alkyl glycerols were 16 and 18 carbon atoms in length,

Giibertson, Johnson, Gelman, and Buffenmyer Aldehydogenic Lipids, Alcohols, and Ethers 497


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other corresponding species present in the l-O-alk-1- enyl glycerols were not observed in the 1-0-alkyl glycerols. This investigation was supported by Public Health Service Research Grant HE 08642 from the Heart and Lung Institute, National Institutes of Health, US. Public Health Service.

Mawcrip t received 14 October 1971; accepted 28 March 1972.

J?’

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natural occurrence of free fattv aldehydes in bovine ... · natural occurrence of free fattv...

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