FATTY ACEE23 {E‘é NEUTRAL MEWS AND PHQSPHQLEPB9$ FROM CHECKEN TISSUES Thesis ‘30? ”w Degree 0? M. S. MICHIGAN STATE UNIVERSITY Michael Alexander Katz 1965 THESIS [J LIBRARY Michigan State Universny ABSTRACT FATTY ACIDS IN NEUTRAL LIPIDS AND PHOSPHOLIPIDS FROM CHICKEN TISSUES by Michael Alexander Katz Lipids from skin, depot fat, dark and white meat from broiler type male chickens were fractionated into neutral and phospholipids by column chro- matography. Lipid fractions were measured gravimetrically as a percentage of total lipids. The fatty acids of each fraction were methylated and ana- lyzed using gas-liquid chromatography. Fatty acid content was calculated as a percentage of the total fatty acids in a fraction. Lipids in muscle tissues were found to contain relatively large quan- tities of phospholipids, while lipids in skin and depot fat contained small amounts. The phospholipid content of dark meat, white meat, skin, and depot fat, was 2:1, 48, 2, and 0.9 percent respectively. The neutral lipid fraction contained at least 18 different fatty acids. The percentage distribution of the fatty acids in the neutral lipids was sim- ilar in all four tissues. Palmitic, palmitoleic, stearic, oleic, and linoleic acicbdominated the fatty acids in the neutral lipids, and amounted to 94 per- cent of the total. The phospholipid fraction contained at least 22 fatty acids. The pre- dominant acids in this fraction were palmitic, stearic, oleic, linoleic, and arachidonic acid. These acids accounted for about 75 percent of the total Michael Alexander Katz fatty acids. The total fatty acid content of the phospholipids varied among the different tissues. Generally, the dark and white muscle tissues had a similar fatty acid composition, which was different from that of skin and depot fat in the content of 020 to 024 fatty acids. The percentage of total molecules of unsaturated fatty acids was ap- proximately the same in the lipids from the four tissues examined. How- ever, the phospholipids contained more long chain fatty acids with four or more double bond, and therefore had a higher degree of unsaturation than the neutral lipids. FATTY ACIDS IN NEUTRAL LIPIDS AND PHOSPHOLIPIDS FROM CHICKEN TISSUES By Michael Alexander Katz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1965 ACKNOWLEDGMENTS My sincere gratitude is extended to: Dr. L. E. Dawson for his guid- ance , advice , and encouragement throughout my graduate program and the preparation of this dissertation; Dr. L. R. Dugan, Jr. for his helpful dis- cussions and criticism; Dr. C. C. Slater for his review of this manuscript; Mr. C. Y. Peng for his suggestions; Mrs. M. Ritchey for assisting in typing; Dr. B. S. Schweigert, chairman, and the faculty of the Department of Food Science, for sharing their knowledge and time with me. Michael Alexander Katz ii TABLE OF CONTENTS ACKNOWLEDGMENTS ....................... LIST OF TABLES .......................... LIST OF APPENDICES INTRODUCTION .......................... LITERATURE REVIEW ........................ Chicken Fat ......................... Lipid Extraction ....................... Fractionation of Neutral and Phospholipids ......... Methyl Esterification of Fatty Acids ............. Chromatography ....................... A. Adsorption chromatography ............. B. Partition chromatography .............. EXPERIMENTAL PROCEDURE .................... Meat Preparation ....................... Lipid Extraction ....................... Separation of Neutral Lipids from Phospholipids ....... A. Preparation and packing of column ......... B. Neutral and phospholipid fraction .......... C. Spot tests used in the fractionation of the lipids . . . D. Percent neutral and phospholipids .......... Preparation of Methyl Esters ................. Gas-liquid Chromatography ................. A. Preparation of column ................ B. Calculation ..................... C. Identification .................... RESULTS AND DISCUSSION .................... Total, Neutral and Phospholipids .............. Fatty Acids from Neutral Lipids ............... Fatty Acids from the Phospholipids .............. Total Unsaturation ...................... SUMMARY AND CONCLUSIONS .................. REFERENCES ............................ APPENDICES ............................ iii Page ii iv VQOUU'IDOOOOH OD I—‘I—‘l—‘l—‘l—‘l—‘I—‘l—‘l—HI—‘t—‘I—‘H \lGGOWQrBwWNNF—‘l—‘H NNNi—‘H .bNNoooo N 03 CON oooo LIST OF TABLES Table Page 1. Total lipid, phospholipid, and neutral lipid content of poultry tissues .................. 18 2. Fatty acids of the neutral lipids .............. 20 3. Fatty acids of the phospholipids .............. 21 4. ' Degree and type of unsaturation of the fatty acids in poultry tissues ................... 25 iv Appendix A. Table Table Table Table Table Table Table Table Table Table Table Figure LIST OF APPENDICES The main fatty acids of the phospholipids and the neutral lipids in the diet ....... Composition of ration fed to broilers used in experiment .............. Fatty acids Fatty acids Fatty acids Fatty acids Fatty acids Fatty acids Fatty acids Fatty acids of the neutral lipids in dark meat . of the neutral lipids in white meat of the neutral lipids in skin. . . . of the neutral lipids in depot fat . of the phospholipids in dark meat. ofthe phospholipids in white meat . of the phospholipids in skin of the phospholipids in depot fat . Percentage of phospholipids found in broiler tissues ................. (a) Series of columns with control valve (b) Valve detail ................ Page 34 35 36 37 38 39 4O 41 42 43 44 45 INTRODUCTION Poultry meat consumption in the United States has increased from 24. 7 pounds per capita in 1950, to over 38. 2 pounds in 1964 (2). A large por— tion of poultry meat has been utilized as boneless meat, processed meat, or in commercial freeze dried products. Since the processed products may be used in combination with other foods and may require a longer shelf life than fresh meat, the needs for detailed compositional data of the meat have increased to aid preservation. The main problem in processed and stored meat is flavor stability. Ox- idative rancidity is a major cause for flavor deterioration. Therefore, the lipids present in fatty tissues as well as lipids present in muscle tissues, may affect flavor quality, and be responsible for problems related to product utilization and stability. The susceptability of natural fat to oxidative rancidity depends largely upon its degree of unsaturation and its fatty acid composition. Fatty acid composition and metabolism have received considerable research attention, although major emphasis has been on problems related to laying hens, eggs, body fluids and organs, and depot fat. Detailed fatty acid analysis of broiler muscle tissues has received little research attention. The great variety of fatty acids present in meat makes a detailed anal- ysis difficult. Many of the fatty acids are present in small quantities, and being homologous compounds, they do not separate readily. For such related compounds, chromatographic analysis has become a suitable means for separation and identification. Gas chromatography, a recent and sensitive tool in this area, enables rapid identification and quantification of fatty acids. The objective of this research was to determine the actual amounts and percentages of fatty acids present in the neutral and phospholipid frac- tions from lipids found in chicken skin, muscle, and depot fat. LITERATURE REVIEW Chicken Fat: In general, animal fats may contain as many as 35 different fatty acids, however, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acids comprise 90 percent of the total amounts present (26). Fats from different animal species differ in their rate of oxidative rancidity. Unsaturated fatty acids tend to oxidize faster than saturated acids (39), and the susceptability of fat to rancidity is therefore related to its fatty acids composition. Poultry fat is more unsaturated than beef, lamb and pork fat (5), and has a total unsaturation of 60-70 percent (5, 19, 20). Because of its un- saturated nature, poultry meat tends to become rancid faster than beef or lamb. Dietary fat affects and reflects the composition of body fat in the chicken (16, 35), and generally body tissues tend to assume the fatty acid composition of the fat in the diet (39). Triglycerides in blood plasma and adipose tissue reflect more closely dietary fatty acids than do the phospho- lipid fractions (27). Many fatty acids can be synthesized in the body, and polyunsaturated acids such as arachidonic, originate from linoleic and linolenic in mammals (55). Linoleic acid is considered to be an essential fatty acid for chickens, and is involved in the synthesis of arachidonic acid (35, 34, 48). Linolenic acid is involved in the synthesis of several polyunsaturated fatty acids (48). Oxidative rancidity of fat is influenced greatly by the unsaturated fatty 4 acids and natural antioxidants present. Phospholipids were reported to oxidize in freeze dried beef prior to neutral lipids (13). The phospholipids contain more unsaturated fatty acids than neutral lipids , and their presence renders lipids more susceptible to oxidation, which is the major factor in deteriorative reactions leading to flavor degradation (33, 56). The majority of arachidonic acid in chicken muscle was found in the phospholipids (44) , thus suggesting this fraction to be an important source of rancidity. Re— search has shown that more long chain polyunsaturated fatty acids are found in muscle than in skin or adipose tissues (39). Depot fat was reported to be similar in composition to body fat in chick- ens, but different in other animals (49, 8). Recently, differences in com- position of muscle tissues from skin and adipose fat were reported (39). However, since skin and adipose fat are low in phospholipids and polyun- saturated fatty acids (56, 27) , compositional difference between these tis- sues and muscle tissue may be quantitative rather than qualitative. Lipid fractionation, therefore, is important for the detection of polyunsaturated fatty acids which are present mainly in the phospholipid fraction. Only a trace amount of these acids is found in unfractionated poultry fat (44). Lipid Extraction: Different solvents can be used to extract lipids from a tissue. Com- parative investigations of different solvent systems for lipid extraction (10) showed that the method in which total lipids are extracted with chloroform and methanol 2:1 (v/v) is preferred (15). The removal of non-lipid material is accomplished by adding water to a final volume of 8:4:3 chloroform-methanol- water. The same solvent system, with different proportions, was used for a rapid lipid extraction which involves a mild and short treatment to mini- mize oxidative decomposition (3). Fractionation of Neutral Phospholipids: Phospholipids are usually more satisfactorily adsorbed on adsorbents than are neutral lipids and fatty acids. The problem, however, was to find an adsorbent from which it is possible to elute the phospholipids quantita- tively (4). After investigating several adsorbents, silicic acid was found to be "the best one " (4). Neutral lipids and free fatty acids can be eluted from a silicic acid column with chloroform, while the phospholipids are ad- sorbed on the column at the rate of 30 mg/g of silicic acid. The phospho— lipids are then eluted quantitatively with methanol. Many workers since have used silicic acid for the fractionation of neutral and phospholipids (51, 9, 13, 17, 32). This separation is possible because of polarity differences of eluting solvents. The polarity of chloro- form can be increased by mixing with methanol or water to further fractionate phospholipids (51). Non—lipid material present in the lipid fraction, is not eluted from the silicic acid column with chloroform or methanol. Such impurities may con— sist of pigments , oxidation products , or denatured protein material, and can be eluted with acetone (32, 13, 25, 9). Although ninhydrin is used mainly for the detection of protein material, it can also be used to detect certain phospholipids (6). Ninhydrin test was used extensively in the detection of phospholipids such as phosphatidyl and lysophosphatidyl ethanolamine, phosphatidyl and lysophosphatidyl serine, sphingomyelin, and others (9, 32, 52, 51, 46, 6). MethLl Esterification of FattxAcids: This procedure involves the liberation of fatty acids from the lipids by saponification, acid hydrolysis, or enzymatic hydrolysis. Esters of the fatty acids may then be prepared by a variety of methods which require acid catalysis of the esterification reaction (41). The chief reaction of fatty acids with sulfuric acid involves the formation of acylium ion-sulfuric acid complex. Acylium ion is then combined with methanol to form methyl esters. Saponification of lipids followed by methyl esterification with acidified methanol was reported with various modifications (36, 21, 28). Acid hy- drolysis of fats and their methyl esterification was accomplished by refluxing with dry HCl methanol solution (12, 28, 54). Hydrolysis of fats with H2804 and esterification with methanol HCl solution on a basic ion exchange resin was also reported (24). A rapid method for the hydrolysis and esterification of fatty acids with excess H2804 at low temperature was described recently (41). Methylation of fatty acids has also been accomplished with boron tri- fluoride—methanol solution (43), and with diazomethane (53). Direct interesterification of triglycerides with methanol in ether con-v taining KOH was also reported (28). Chromatography: Chromatography is an analytical procedure frequently used for the sep- aration of closely related compounds. Fundamentally the technique uses a two phase system: a stationary phase and a mobile phase. The stationary phase may be a solid (adsorption chromatography) or a liquid (partition chro— matography) (50). Adsorption chromatography includes column and thin layer chromatography, and partition chromatography includes gas-liquid chroma- tography. One advantage of gas chromatography over other analytical pro- cedures is the rapidity of identification and quantification, which are ac— complished simultaneously. A. Adsorption chromatography: Adsorption chromatography consists of a stationary solid (adsorbent) and a mobile liquid. In column chromatography, the solid is held in a tube, while the liquid moves past it carrying the sample. Thin layer chromatog- raphy is essentially an open column chromatography, in which the solid stationary phase is coated over glass plates. The adsorbents used in adsorption chromatography adsorb polar com— pounds to their surface by electrostatic forces (the same forces which hold the crystal lattice together). These electrical surface forces induce dipole moment in non-polar compounds , and increase the existing dipole moments of the polar compound (47). The rate of migration of a compound on a given adsorbent, depends upon the solvent used. Solvents have different elutive power (47) and polar solvents have greater elution power than non-polar sol- vents (38). A mixture of solvents gives a better separation than one solvent (38, 47). The detection of a compound separated by adsorption chromatography is accomplished colorimetrically by special applications during the devel- oping (migration) stage. Fractions resolved by adsorption chromatography may be isolated and further analyzed by complementary methods such as gas-liquid chromatography (GLC). B. Partition chromatography: Partition chromatography is accomplished by partition between two im- miscible liquids , one of which is held stationary while the other liquid moves past it. In gas-liquid chromatography, the mobile phase is an inert gas which is compressible to produce a gradient of gas velocity down the column. The sorbent is a non-volatile liquid coated on a finely divided inert solid support which is enclosed in a column. The sample is introduced as a nar- row band of vapor into the continuous stream of carrier gas. The volatilized sample is distributed between the moving gas and the stationary liquid, and the individual components move down the column at a rate which depends on their partition coefficient. Stationary phases differ in holding capacity (or retention value) of the dissolved sample molecules, and those which hold the dissolved molecules by Van de Waals forces (induced dipoles) have a lower retention value than those permitting hydrogen bonding (50). The rela— tive retention value of homologous series give a linear relation when plotted, whose slope depends on the amount of hydrogen bonding (29). The various components of the sample are detected by passing the carrier gas, with the eluted sample, through a suitable flame ionization detector. Detection is based on the measurement of electrical conductivity of gases in hydrogen flame. The conductivity of hydrogen burning in air is low, but , when an organic substance is fed into the flame , the conductivity increases (23). The detector is connected to a recorder pen which records, by deflec- tion, peaks corresponding to concentration of the eluate over its retention time. Since retention value is specific to a substance, both quality and quantity are recorded simultaneously. Gas-liquid chromatography is a most valuable tool for the analysis of fatty acids. The theory of gas chromatography was introduced in 1952 (29) as a method for separating carboxylic acids up to 12 carbon atoms. Later the method was extended to the separation of methyl esters of fatty acids (7). Methyl esters have lower boiling points than fatty acids, thus they vaporize at lower temperatures and minimize the danger of decomposition (45). Today GLC has been adapted for use in many diversified areas. Initially non—specific liquid phases, such as silicone oil or apiezon L greases, were used for column separation. With these liquid phases it is difficult to separate isomers with the same carbon number. The polyester type liquid phases gave the necessary separation efficiency. Of these types, adipate and succinate polyesters of diethelene glycol are the most widely used (14). The addition of phosphoric acid to the liquid phase proved to be useful in retaining symmetrical peaks for long chain fatty acids (42). Retention value in GLC depends on the type and amount of liquid phase, temperature, gas flow, and type of compound analyzed (45). Satisfactory application of GLC to fatty acid analysis , however, depends also on care- ful control of temperature and sample size (22) , making it a delicate operation. 10 The measurement of peak area gives a direct weight percentage meas— urement for methyl esters of fatty acids with over eight carbon atoms (22, 14). Several ways to measure the peak area have been suggested (22), but using the integrator recordings for the measurement is simplest. Identification of the different peaks of a chromatograph can be accom- plished by comparing their retention value to that of a standard. Since all standards are not readily available, a standard curve can be plotted, in which the log retention time is plotted against carbon number (28). This method of identifying components is widely used. A more accurate identi- fication is achieved by using different column packings, and by plotting the log retention time of the standards on a polar column vs the log reten- tion time on a non—polar column (28). On both curves, homologous com— pounds with the same degree of saturation form a linear relation. Some compounds may have the same retention value at a given operating condition, thus appearing in a common peak. These compounds may be re- solved by programming the temperature, using different isothermal tempera- ture (l), and using new and old columns (37). EXPERIME NTAL PROCE DURE Meat Preparation: Twelve 14 weeks old male broilers obtained from a commercial farm were killed, defeathered and eviscerated in the usual manner, and chilled in crushed ice for four hours. From this group, four uniform birds weighing 2 kg each were selected for analysis. Muscle samples (100 g each) were removed from the corresponding one-half of each carcass. Samples of white meat were taken from the breast muscles and dark meat samples were taken from the thigh and drumstick. Fifty g of skin were used for the skin sample, and all removable depot fat from the abdominal cavity, after the removal of the viscera, was used for depot fat sample. Muscles and skin tissues were ground prior to lipid extraction. Lipid Extraction: Total lipids were extracted with chloroform~methanol-water solution 8:4:3 (V/V/V) (l5, 3). A 100g ground sample (76 percent water content) was blended in a Waring Blender for two minutes with 100 ml chloroform, 200 ml methanol, and 4 ml water. One hundred ml chloroform was added and the mixture blended for 30 minutes after which 100 ml water was added and the mixture blended for an additional 30 minutes. The resulting mixture was then filtered on a Buchner funnel, and the residue plus the filter paper was blended for one minute in 100 ml chloroform, filtered, and rinsed with 180 ml chloro- form and 40 ml methanol. The filtrates were collected in a separatory funnel and held at 30°C 11 12 overnight to facilitate separation. A biphase liquid system was formed. The upper phase of water and methanol was discarded, and the lower phase, containing the lipids in chloroform and some methanol, was saved for anal- ysis. Seflration of Neutral Lipids from Phosgholipids: Fractionation of the lipids was accomplished on a silicic acid column (4). A multiple column system (31) was used, where four columns were connected in a series to a nitrogen source by a rubber hose. A special valve, which eliminated high pressure buildup in the column, was attached to the rubber hose (see Appendix E). A. Preparation and packing of the column: Silicic acid (80-100 mesh) was washed with distilled water to remove the fines. Water was then removed from the silicic acid on a Buchner fun- nel, and this was followed by a methanol rinse. The washed silicic acid was activated by holding overnight at 1050C. A 10 g sample of activated silicic acid-chloroform slurry was poured into a glass column (1 cm in diameter), fitted with a glass wool stopper in its lower end. The silicic acid was allowed to settle and the chloroform drained under slight nitrogen pressure. After the silicic acid settled, a 2-3 cm layer of granular anhydrous sodium sulfate was placed on top of the silicic acid. The column was rinsed several times with chloroform and was ready for use. 13 B. Neutral and phospholipid fractions: A solvent column containing about 0. 2 g crude lipids was placed in the column. The sample was eluted from the column under nitrogen pressure, at the rate of two drops per second, into drying flasks. The chloroform eluant of the sample was taken as neutral lipids, and the methanol eluant as phospholipids. Neutral lipids were eluted until a negative Salkowski test was achieved, and phospholipids were eluted until a negative ninhydrin test was reached. Purity was further tested by checking the neutral lipids with ninhydrin, and the phospholipids by micro thin layer chromatography (TLC). New column packing material was used for each sample, and the non-lipid material present in the lipid fraction was discarded. The neutral and phospholipid fractions were then prepared for fatty acids esterification, or dried for the gravimetric determination of total neu- tral and phospholipids . C. Spot tests used in the fractionation of the lipids: Salkowski test. A sample dissolved in chloroform was carefully added to an equal volume of concentrated sulfuric acid in a test tube. The devel— opment of a characteristic yellow band in the test tube indicated the presence of lipids in the chloroform. Ninhydrin test. Ninhydrin was added to the methanol eluant in a test tube, placed in a sand bath at 150°C, and shaken vigorously periodically. The development of a bluish color indicated the presence of phospholipids. Micro TLC. Micro slides (2. 5 x 7 cm) were covered with adsorbent by dipping them into a suspension of silica gel G in chloroform. The chromoslides WE 1( ct co ev fla nig phc CO in Fifi met 14 were allowed to dry, spotted with phospholipid fraction, and eluted in an ascending manner with petroleum ether, ethyl ether, and acetic acid (90: 10:1 by volume). The slides were sprayed with sulfuric acid followed by charring. Migration of the original spot or streaking, indicated presence of neutral lipids . D. Percent of neutral and phospholipids: A known quantity of crude lipids was fractionated on a silicic acid column. The fractions were evaporated in tared flasks on a Rinco vacuum evaporator at 50°C, and dried for 10 minutes in an oven at 110°C. The flasks were then placed in a dessicator and allowed to equilibrate over- night, and then weighed. The combined weight of the dried neutral and phospholipids was taken as 100 percent of the lipid content. Preparation of MethLl Esters: Fatty acids were esterified at low temperature (41). A solvent volume containing not more than 200 mg neutral or phospholipids was concentrated in a flask on a Rinco vacuum evaporator in a 40°C water bath. The lipids were dissolved in 20 ml ethyl ether and the flask placed in a dry ice acetone bath on a magnetic stirrer. When the dissolved lipids reached the bath tem- perature, 2 ml of concentrated H2804 were added dropwise to the stirred sample. The flask was then corked and stirred for 10 minutes in the bath. Fifteen ml of absolute methanol was added followed by 22 m1 of 25 percent methanolic KOH. The mixture was then removed from the bath, phenolpthalein added to ascertain the presence of enough base, and stirred to red color. 15 The mixture was quantitatively transferred to a 250 ml separatory funnel with 200 ml distilled water and the fatty acids extracted with 20 and 15 ml petroleum ether (30-600C B.P. ). The ether extracts were dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in a 15 m1 grad- uated centrifuge tube over 60°C water bath, under a nitrogen atmosphere. Neutral lipid solutions were concentrated to 0. 2 ml, and phospholipid solu- tion to 0.1 ml. The concentrated solutions of methyl esters of fatty acids were trans- ferred, with a syringe, to small tubes, made from No. 4 glass tubing by heat sealing one end, and stoppered with parafilm. These samples were kept at 0°C in the dark for not more than 12 hours prior to GLC analysis. The completeness of methyl esterification was checked by TLC. Chro— matographic plates were prepared with a Desaga applicator by making a slurry of 25 g silica gel G with 50 ml distilled water. The chromoplates were activated at 1100C for 35 minutes. Samples were spotted and devel- oped in an ascending manner in petroleum ether—ethyl ether-glacial acetic acid solution 90:10:1 (by volume). The plates were sprayed with sulfuric acid and charred to visualize the methyl esters and non—esterified material. Gas—liquid Chromatograply: Fatty acid methyl esters were fractionated in an F & M (Model 810) dual column gas chromatograph, equipped with a flame ionization detector and a disc chart integrator. Helium was used as the carrier gas at a flow rate of 35 ml/min. The hydrogen flame was fed by using 60 ml/min hydro- gen and 170 ml/min compressed air. A 72 x 1/4" copper column was packed 16 with 15 percent diethylene glycol succinate and 3 percent phosphoric acid as liquid phase and chromosorb—W as solid support. The column tempera— ture was run isothermally at 190°C, with detector temperature at 260°C and injector at 250°C. Attenuation was 102 x 32 for neutral lipids and 102 x 4 for phospholipids . A. Preparation of column: Three 9 of phosphoric acid were dissolved in distilled water and mixed with 32 g acid washed, 80-100 mesh, chromosorb--W. The compounds were mixed in a round bottom flask, on a Rinco rotator, and dried in an oven. Fifteen g of diethylene glycol succinate (DEGS) dissolved in chloroform were added to the dried mixture and enough chloroform to assure proper mixing was added. After mixing and evaporation on the Rinco evaporator, the residual chloroform was removed in an air oven, and the dried mixture was ready for packing. Copper column tubings were packed with the aid of an electrical vi- brator. Glass wool plugs were placed in both ends of the column to pre- vent loss of packing. The packed columns were conditioned for two days at 220°C and 35 ml/min helium flow before using. B. Calculation: Percentage composition of the fatty acids was calculated from the peak area measurement associated with each fatty acid. Peak area was calcu- lated from the pen traces made under the peak by the disc chart integrator, taking the total tracings as 100 percent. 17 Two isothermal runs at different sensitivities were made, and the final results were calculated with a reference to methyl myristate which was well defined in each run. C. Identification: Methyl esters ofC 8:0,110:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:4, 20:5, 22:0, and 2226 were identified by com— paring the retention time to that of pure samples. Using the above standards , the log of retention time was plotted against carbon number to give a stand- ard curve which was used in the identification of other fatty acids. Identification of the extremely short and long fatty acids was aided with programming, use of old and new columns at the same operating conditions, use of the same column at different temperatures, and the use of a non-polar apiezon L column. 1The first number indicates the carbon chain length. The colon is followed by the number of double bonds per molecule, if any (11). RESULTS AND DISCUSSION Total, Neutral, and Phospholipids: The percentage of total lipids in dark and white meat, skin, and depot fat varied considerably as expected. Total lipids varied on a wet weight basis from 1 percent in white meat to 60-80 percent in depot fat, as shown in Table 1. In agreement with other work (5) dark meat had twice as much lipid as did white meat. Generally, the lipid content of skin varied with the apparent amount of fat deposited. Table 1. Total lipid, phospholipid, and neutral lipid content of poultry tissues Lipids Tissue Totall Phospholipids2 Neutral2 percent White meat 1. 0 48 52 Dark meat 2. 5 21 79 Skin 25 2 98 Depot fat 60-80 0. 9 99.1 lAs percentage of raw tissue. 2 As percentage of total lipids. Separation of the lipids into neutral and phospholipids, revealed ad" ditional differences among tissues (Table 1). White meat, the lowest in total lipids, contained almost equal amounts of neutral and phospholipids. As the total lipid content in tissues increased, the ratio of neutral to 18 19 phospholipids increased. Depot fat tissue, the richest in total lipids, con-— tained 99 percent neutral lipids and only 1 percent phospholipids. These results are in general agreement with other reports. A large content of phospholipids in skeletal muscle of birds was reported (9 , 17) where lipids of pigeon breast muscle contained over 40 percent phospholipids. Lipids of adipose tissue in hens were reported to have only 2 percent phospholipids (27), and skin lipids were reported to be similar to abdominal adipose tis- sue (40). Many workers (8, 16, 20, 36) who studied the composition of fatty acids in chicken tissues , concentrated their investigations on fat rich tis- sues such as skin and depot fat. These tissues contain relatively small amounts of phospholipids , and are therefore relatively low in polyunsaturated acids. Due to such minute quantities present, it is probable that the long chain polyunsaturated fatty acids , above arachidonic acid, were not de- tected, and thus not reported. The presence of long chain polyunsaturated fatty acids in muscle tissue lipids, and their absence in skin and depot fat has been reported (39). However, this observation may have been af- fected by the high concentration of phospholipids in muscle tissues and the low concentration of these lipids in skin and depot fats as shown in Table 1. For a detailed study of polyunsaturated fatty acids in poultry tis- sues, the phospholipids should be fractionated before analysis, since more of the polyunsaturated fatty acids and those with carbon number greater than C20, are present in this fraction, as shown in Tables 2 and 3. The low level of the phospholipids and consequently of some of the long chain 20 polyunsaturated fatty acids in poultry tissues which are rich in fat, may account for the apparent lack of compositional changes in lipids from these tissues, due to oxidative deterioration (5, 10). It is doubtful that an ex— amination of the unfractionated lipids could detect compositional changes. Table 2. Fatty acids of the neutral lipids1 Source of fatty acids Fatty2 Dark White Depot acid meat meat Skin fat percent 8:0 tr tr tr tr 10:0 tr tr tr tr 12:0 tr tr tr tr 14:0 0.8 1.0 0.7 0.7 14:1 0.3 0.3 0.3 0.2 15:0 0.2 0.2 0.2 0.2 16:0 22.4 24.2 23.1 22.8 16:1 6.7 5.5 5.7 5.7 16:2 0.5 0.5 0.4 0.5 17:0 0.2 0.3 0.2 0.2 18:0 6.4 5 9 5.9 6.5 18:1 34.6 34.6 37 0 37.0 18:2 24.7 24.7 24.0 23.7 18:3 1.5 1.3 1.3 1.3 20:1 0.5 0.5 0.6 0.6 20:2 0.3 0.2 0.2 0.2 20:3 0.2 0.3 0.1 0.1 20:4 0.5 0.5 0.2 0.2 1Calculated as percentage of the total fatty acids in the neutral lipids. 2Number of carbons : number of double bonds. 21 Table 3. Fatty acids of the phospholipids1 2 Source of fattxacids Fatty Dark White Depot acid meat meat Skin fat percent 10:0 tr tr tr tr 12:0 tr tr tr tr 14:0 3.2 0.1 3.65 1.8 14:1 0.3 0.1 0.5 1.1 15:0 0.7 0.3 0.7 0.8 16:0 14.6 23.0 22.1 18.7 16:1 1.6 0.9 3.1 5.0 16:2 0.5 0.3 0.7 0.7 17:0 0.4 0.4 1.0 0.6 18:0 16.6 9.8 11.8 9.0 18:1 13.5 16.3 18.5 25.5 18:2 19.8 17.0 14.8 22.3 18:3 0.6 0.5 1.0 1.7 20:1 0.6 0.6 0.8 1.2 20:2 0.8 0.8 0.9 0.7 20:3 1.2 1.5 1.6 0.6 20:4 16.8 15.1 11.4 4.6 22:2 0.4 0.7 1.1 0.9 22:3 0.3 0.7 0.6 0.7 22:4 2.5 3.0 2.7 1.3 24:1 0.9 1.0 1.7 1.6 22:5-24:2 1.5 1.7 0.9 0.2 22:6-24:4 3.3 3.9 1.4 1.3 2Number of carbons 1Calculated as percentage of total fatty acids in the phospholipids. : number of double bonds. 22 Fatty Acids from Neutral Lipids: The fatty acid composition of the neutral lipid fractions from the dif— ferent tissues was calculated as a percentage of the total fatty acids in these fractions. Results are shown in Table 2. Eighteen different fatty acids were identified and quantified from the neutral lipid fraction from dark and white meat, skin, and depot fat tissues. The similarity in the relative amount of the fatty acids found in neutral lipids from these four tissues is of major significance. Such similarity in the triglyceride con- tent has been previously reported (17). The most prevalent fatty acids in these fractions had 16 carbon atoms (palmitic and palmitoleic), and 18 carbon atoms (stearic, oleic, and linoleic acid). These C16 and 018 fatty acids amounted to 94 percent of the total fatty acids in the neutral lipids. Palmitic acid accounted for a major portion of the saturated fatty acids (about 23 percent out of 30 percent). A large percentage of the unsaturated acids were oleic and linoleic acids which amounted to about 60 percent. This distribution of fatty acids is quite typical and was reported by other workers (27 , 26). Saturated fatty acids with odd numbers of carbon atoms were minor components in all. the tissues, and their presence was previously reported in depot fat (35 , 27). However, since the identification of these acids was established indirectly by a comparison to the standard plot, these odd carbon peaks possibly may correspond to dimethylacetals of aldehydes of other fatty acids. Fatty Acids from the Phospholipids: Twenty-two fatty acids were identified and quantified in the phospholipid 23 fraction from the lipids obtained from the dark and white meat, skin and depot fat of broilers. The predominant fatty acids in this fraction were palmitic, stearic, oleic, linoleic, and arachidonic acids. These acids accounted for about 75 percent of the total fatty acids. The fatty acid content of the phospholipids varied among the different tissues. Although fatty acid composition from the white and dark meat phospholipids were similar, they differed from that found in skin and depot fat. The composi— tion of fatty acids from skin phospholipids , resembled phospholipids from depot fat more than the phospholipids from muscle tissue. The most apparent difference in the phospholipid fatty acid composi- tion of the different tissues was a variation in the arachidonic acid content. The percentage of arachidonic was lower in the lipid rich tissues (skin and depot fat) than in the muscle lipids: only 4 percent arachidonic acid was found in the depot fat as compared to 16 percent in the dark muscle meat. Phospholipids from dark meat contained the lowest percentage of palmitic, and the highest percentage of stearic and arachidonic acids. Phospholipids from the white meat lipids were low in palmitoleic, high in arachidonic, and contained equal amounts of oleic and linoleic acids. Depot fat phos- pholipids were highest in palmitoleic, oleic, and linoleic, and lowest in arachidonic acid. The fatty acid composition from the skin phospholipids resembled those from depot fat, with the exception of arachidonic acid. All tissues examined contained small amounts of saturated fatty acids with 15 and 17 carbons. Such acids were previously found in phospholipids of pigeon (17), and chicken (44, 27). Detailed comparative quantitative 24 investigation of total fatty acids in the neutral lipids and the phospholipids from poultry tissues has not been reported. However, similar work on total fatty acids in the phospholipids confirmed the presence of the acids reported here. Investigation of the fatty acids in specific phospholipids (17, 46) indicated that arachidonic acid is a major component of several phospho— lipids, and that 19. 2 and 16. 3 percent arachidonic acid was present in the phospholipid fraction of pork and beef, respectively (25). Fatty acids from the phospholipids had a larger variation in their rela- tive distribution in the same tissue of different birds ,. than those from the neutral lipids. These differences are due to biological variability and are shown in Appendix C. Similar differences were observed previously (18, 25). Total Unsaturation: The weight percentage of total molecules of unsaturated fatty acids was approximately the same in the lipids from the four tissues examined, as shown in Table 4. The fatty acids in the neutral lipids had only slightly more unsaturated molecules than the fatty acids in the phospholipids. Similar fatty acid characteristics were found in plasma and adipose lipids of laying hens (27). However, the phospholipids contained more long chain fatty acids with four or more double bonds than the neutral lipids. These fatty acids make the phospholipids more susceptible to oxidative deterioration, which is more severe in polyunsaturated than in saturated fats (33, 56). The results obtained in this experiment support previous suggestion 25 that flavor deterioration may result from fat oxidation in muscle tissue (39) , and not only from oxidation in fatty tissues. The relatively large quantities of highly unsaturated fatty acids, helps to provide an explanation for the relative ease with which oxidative rancidity occurs in poultry meat. Based on the polyunsaturated fatty acids present in the phospholipids , muscle tis~ sues have the same potential for oxidation as does depot fat. Table 4. Degree and type of unsaturation of the fatty acids in poultry tissues No. of Source of fatty acids double Dark White Depot bonds meat meat Skin fat a.) Fatty acids of the phospholipids Weight percent 0 34.5 34.6 37.3 30.2 1 16.9 19.0 25.1 32.8 2 21.4 19.1 17.4 23.9 3 2.1 2. 1 3.2 2.6 4 19.8 18.3 14.1 5.9 5 1.5 1.7 0.9 0.3 6 3.3 3.9 1.4 1.3 0 (odd C#) 1.0 1.3 1.7 1.5 Unsaturated fatty acids 64.5 64.1 61.0 68.2 b.) Fatty acids of the neutral lipids Weight percent 0 28.9 31.2 29.7 30.1 1 42.2 40.1 43.5 43.6 2 25.5 25.5 24.7 24.4 3 1.7 1.6 1.5 1.4 4 0.5 0.5 0.2 0.2 0 (odd C#) 0.4 0.5 0.3 0.3 Unsaturated fatty acids 70.7 68.3 69.0 69.3 SUMMARY AND CONCLUSIONS Lipids from poultry skin, depot fat, and muscle tissues, contain es- sentially the same fatty acids. Differences in the fatty acid content are quantitative rather than qualitative, and are due to the variation in total lipids and phospholipids content of the tissues. The lipids from muscle tissues contained a large percentage of phos-- pholipids, while lipids from skin and depot fat contained a small percent- age of phospholipids. At least 22 fatty acids, varying in chain length from C10 to C24, were found in the phospholipid fraction. The predominant acids in this fraction were palmitic, stearic, oleic, linoleic, and arach— idonic acid. Arachidonic acid accounted for the main difference in the fatty acids content of the phospholipids in the tissues examined. The percentage of arachidonic acid was high in muscle tissues , and decreased with the increase of total lipids in depot fat and skin tissues. The relative distribution of the fatty acids in the neutral lipids, were similar percentagewise in all the tissues examined. Eighteen fatty acids , varying in chain length from C8 to C20, were found in this fraction. The predominant fatty acids in this fraction were palmitic, palmitoleic, stearic, oleic, and linoleic acid. About 60 to 70 percent of the fatty acids in poultry tissues were un- saturated. However, based on the number of double bonds present, fatty acids from the phospholipids showed a much higher degree of unsaturation 26 27 than those from the neutral lipids. 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Deposition of polyunsaturated fatty acids in fat deficient rats upon single fatty acid supplementation. Arch. Biochem. 25:1. APPENDICES 33 34 APPENDIX A Table 1. The main fatty acids of the phospholipids and the neutral lipids in the diet Lipid fraction Fatty acid1 Phospholipids Neutral lipids Percent 14:0 0.3 0.3 16:0 20.4 12.4 16:1 0.8 0.7 18:0 3.5 2.9 18:1 15.4 26.9 18:2 49.5 53.6 18:3 9.5 3.0 20:4 0.5 tr. 1Carbon number : number of double bonds. Table 2 . Composition of ration fed to 35 broilers used in experiment1 Age 1-10 10 days 6—10 10-13 13 wks Component days - 6 wks wks wks & up Peter Hand Custom 50 - - -- - Peter Hand FW poultry — 50 50 38 40 Ground yellow corn 1100 1072 1175 1455 1425 Soybean oil meal (50%) 675 475 325 150 2000 Meat & bone scraps (50%) 50 100 100 100 100 Dehydrated alfalfa meal 38 50 50 50 50 Wheat standard midds - 200 250 150 150 Salt 5 8 8 5 8 Dicalcium phosphate 20 23 25 - 18 Ground limestone - 13 10 10 10 Defluorinated phosphate — - - 18 — PH trace mineral mix - 1/2 1/2 - 1/2 CCC trace mineral mix 1 - - 1/2 - Fat 40 - - - — Aurofac 10 20 - — - - Zoamix 1 10 10 10 10 % protein 24 22 19 15. 6 16 1 Expressed as pounds per ton. 36 APPENDIX B Table 1. Fatty acids of the neutral lipids in dark meat Fattyl Bird number acid 1 2 3 4 Average percent of total fatty acids 8:0 tr. tr. tr. tr. tr. 10:0 tr. tr. tr. tr. tr. 12:0 tr. tr. tr. tr. tr. 14:0 0.8 0.8 0.9 0.8 0.8 14:1 0.3 0.3 0.4 0.2 0.3 15:0 0.1 0.2 0.1 0.2 0.2 16:0 22.1 20.7 22.8 24.1 22.4 16:1 7.6 6.9 7.2 5.3 6.7 16:2 0 5 0.6 0.5 0.5 0.5 17:0 0.2 0.2 0.2 0.2 0.2 18:0 4 8 6.3 6.1 8 2 6.4 18:1 37.2 35.8 33.1 32.3 34.6 18:2 24.0 24.2 25.7 24.9 24.7 18:3 1.5 1.6 1.4 1.4 1.5 20:1 0.4 0.8 0.4 0.6 0.5 20:2 0.1 0.4 0.2 0.4 0.3 20:3 0.3 0.3 0.2 0.3 0.3 20:4 0.3 0.7 0.6 0.6 0.5 1Number of carbons : number of double bonds. 37 Table 2. Fatty acids of the neutral lipids in white meat Fatty1 Bird number acid 1 2 3 4 Average percent of total fatty acids 8:0 tr. tr. tr. tr. tr. 10:0 tr. tr. tr. tr. tr. 12:0 tr. tr. tr. tr. tr. 14:0 0.8 1.0 1.0 1.1 1.0 14:1 0.4 0.2 0.3 0.4 0.3 15:0 0.2 0.2 0.2 0.3 0.2 16:0 24.1 23.5 24.3 24.7 24.2 16:1 6.0 5.5 5.0 5.6 5.5 16:2 0.4 0.7 0.5 0.6 0.5 17:0 0.2 0.3 0.3 0.4 0.3 18:0 5.0 6.7 6 4 5.4 5 9 18:1 36.1 35.0 32.7 34.7 34.6 18:2 24.1 24.0 25.9 24.7 24.7 18:3 1.5 1.1 1.5 1.2 1.3 20:1 0.5 0.6 0.5 0.4 0.5 20:2 0.2 0.3 0.2 0.3 0.2 20:3 0.2 0.2 0.3 0.3 0.3 20:4 0.3 0.4 0.6 0.5 0.5 1Number of carbons : number of double bonds. 38 Table 3. Fatty acids of the neutral lipids in skin Fatty1 Bird number acid 1 2 3 4 Average percent of total fatty acids 8:0 tr. tr. tr. tr. tr. 10:0 tr. tr. tr. tr. tr. 12:0 tr. tr. tr. tr. tr. 14:0 0.8 0.5 0.8 0.8 0.7 14:1 0.3 0.1 0.4 0.1 0.2 15:0 0.2 0.1 0.1 0.2 0.2 16:0 22.7 22.9 24.0 22.7 23.1 16:1 7.2 3.8 6.5 5.1 5.7 16:2 0.4 0.4 0.4 0.5 0.4 17:0 0.3 0.1 0 2 0.1 0.2 18:0 4.9 6.4 5 8 6.6 5 9 18:1 37.0 39.4 35 5 35.9 37.0 18:2 23.2 24.4 23.7 24.9 24.0 18:3 1.8 0.9 1.4 1.4 1.3 20:1 0.7 0.4 0.5 0.6 0.5 20:2 0.2 0.1 0.2 0.3 0.2 20:3 0.2 tr. 0.1 0.2 0.1 20:4 0.2 0.1 0.2 0.3 0.2 1Number of carbons : number of double bonds. 39 Table 4. Fatty acids of the neutral lipids in depot fat Fatty Bird number acid 1 2 3 4 Average percent of total fatty acids 8:0 tr. tr. tr. tr. tr. 10:0 tr tr. tr. tr. tr. 12:0 tr tr. tr tr tr. 14:0 0.7 0.7 0.8 0.7 0.7 14:1 0.3 0.2 0.2 0.2 0.2 15:0 0.2 0.1 0.2 0.2 0.2 16:0 21.7 22.5 23.3 23.7 22.8 16:1 7.7 4.0 6.9 4.3 5.7 16:2 0.4 0.5 0.4 0.4 0.4 17:0 0.2 0.2 0.2 0.2 0.2 18:0 6.4 8.2 4.4 7.1 6.5 18:1 37.3 37.5 37.0 36 2 37.0 18:2 22.7 23.2 24.3 24.6 23.7 18:3 1.4 1.1 1.3 1.2 1.3 20:1 0.6 0.7 0.5 0.6 0.6 20:2 0.1 0.3 0.2 0.2 0.2 20:3 tr. 0.1 0.1 0.1 0.1 20:4 0.1 0.2 0.1 0.2 0.1 1Number of carbons : number of double bonds. 4 0 APPENDIX C Table 1. Fatty acids of the phospholipids in dark meat Fattyl Bird number acid 1 2 3 4 Average percent of total fatty acids 10:0 tr. tr. tr. tr. tr. 12:0 tr. tr. tr. tr. tr. 14:0 3.3 2.6 1.9 4.4 3.2 14:1 0.1 0.1 0.1 0.1 0.1 15:0 1.4 0.2 0.6 0.4 0.6 16:0 13.7 13.5 14.6 16.5 14.6 16:1 2.6 1.0 1.1 1.6 1.6 16:2 0.3 0.4 0.3 0.6 0.4 17:0 0.9 0.1 0.4 0.2 0.4 18:0 14.4 19.0 17.5 15.7 16.6 18:1 14.6 10.1 13.5 15.8 13.5 18:2 18:0 20.6 21.7 18.6 19.8 18:3 0.6 0.4 0.7 0 7 0.6 20:1 0.7 0.4 0.4 0.9 0.6 20:2 0.7 0.6 0.6 1.3 0.8 20:3 1.4 0.9 1.2 1.4 1.2 20:4 14.0 19.1 18.6 15.5 16.8 22:2 1.1 0.4 0.1 0.1 0.4 22:3 0.6 0.3 0.2 0.1 0.3 22:4 3.1 2.5 2.5 1.8 2.5 24:1 1.2 1.2 0.6 0.5 0.9 22:5-24:2 2.0 2.0 1.2 0.9 1.5 22:6-24:4 4.1 4.4 2.4 2.4 3.3 1Number of carbons : number of double bonds. 41 Table 2. Fatty acids of the phospholipids in white meat __ Fattyl Bird number acid 1 2 3 4 Average percent of total fatty acids 10:0 tr. tr. tr. tr. tr. 12:0 tr. tr. tr. tr. tr 14:0 2.3 1.9 1.7 1.4 1.8 14:1 2.3 tr. 0.1 tr. 0.1 15:0 1.2 0.6 0.2 1.3 0.8 16:0 24.9 23.5 24.5 18.9 23.0 16:1 1.8 1.0 0.8 0.2 0.9 16:2 0.4 0.3 0.2 0.1 0.3 17:0 0.4 0.4 tr. 0.7 0.4 18:0 8.8 9.3 11.9 9.4 9.8 18:1 19.6 14.5 15.2 15.8 16.3 18:2 17.2 16.4 17.0 17.4 17.0 18:3 0.3 1.3 0.1 0.3 0.5 20:1 0.3 1.8 0.2 0.3 0.6 20:2 0.7 1.1 0.6 0.9 0.8 20:3 1.4 1.4 1.6 1.6 1.5 20:4 12.1 15.1 17.1 15.9 15.0 22:2 0.3 0.7 0.7 1.1 0.7 22:3 0.8 0.7 0.1 1.0 0.7 22:4 3.7 2.1 2.8 3.3 3.0 24:1 0.8 1.0 0.5 1.8 1.0 22:5-24:2 1.0 2.0 1.0 2.6 1.7 22:6-24:4 3.3 4.1 3.4 4.7 3.9 1Number of carbons : number of double bonds. 42 Table 3. Fatty acids of the phospholipids in skin Fatty1 Bird number acid 1 2 3 4 Average percent of total fatty acids 10:0 tr. tr. tr. tr. tr. 12:0 tr. tr. tr. tr. tr. 14:0 3.3 5.0 2.9 2.7 3.0 14:1 0.8 0.3 0.7 0.7 0.5 15:0 0.5 0.7 0.3 1.2 0.7 16:0 24.1 18.7 25.5 19.7 22.0 16:1 4.6 2.8 1.2 3.2 3.1 16:2 0.7 0.9 0.5 0.7 0.7 17:0 tr. 0.6 0.3 2.0 1.0 18:0 11.4 15.4 9.4 10.9 11.8 18:1 16.8 15.7 21.7 20.0 18.5 18:2 17.9 11.0 14.4 15.9 14.8 18:3 1.8 0.6 0.4 1.2 1.0 20:1 1.3 0.6 0.3 1.0 0.8 20:2 0.9 0.7 0.8 1.0 0.9 20:3 2.1 1.3 1.7 1.1 1.6 20:4 9.8 13.9 12.2 9.6 11.4 22:2 0.8 0.6 0.4 2.5 1.1 22:3 tr 0.5 0.3 1.1 0.6 22:4 1.9 3.7 3.4 1.8 2.7 24:1 0.6 3.6 0.6 1.8 1.7 22:5-24:2 O. 6 1. 4 1. 2 0. 5 0. 9 22:6-24:4 1.0 1.7 1.6 1.3 1.4 1Number of carbons : number of double bonds. 43 Table 4. Fatty acids of the phospholipids in depot fat Fatty1 Bird number acid 1 2 3 4 Average percent of total fatty acids 10:0 tr. tr. tr. tr. tr. 12:0 tr. tr. tr. tr. tr. 14:0 21 1 3 1.2 2.7 1.8 14:1 1.4 1.1 0.6 1.1 1.1 15:0 0.8 0.8 0.5 1.2 0.8 16:0 17.7 19.1 21.0 17.0 18.7 16:1 7.3 3.0 5.8 3.8 5.0 16:2 0.7 tr. 0.6 0.7 0.7 17:0 0.4 0.9 0.6 0.6 0.6 18:0 10.9 6.5 8.7 9.8 9.0 18:1 21.2 29.2 28.6 23.0 25.5 18:2 25.4 20.3 22.4 20.1 22.3 18:3 1.4 1.7 1.6 2.0 1.7 20:1 1.0 1.3 0.9 1.4 1.8 20:2 0.5 0.9 0.7 0.7 0.7 20:3 0.6 0.2 0.5 1.2 0.6 20:4 3.1 5.7 3.6 5.0 4.6 22:2 0.8 1.0 0.5 1.8 0.9 22:3 0.6 1.3 tr. 0.9 0.7 22:4 0.9 1.5 0.8 1.7 1.3 24:1 1.2 2.0 1.0 2.3 1.6 22:5-24:2 tr. 0.2 1.0 0.4 0.2 22:6-24:4 1.2 1.4 0.6 1.9 1.3 1Number of carbons : number of double bonds. 44 APPENDIX D Table 1. Percentage of phospholipids found in broiler tissues ' Source of phospholipids Bird No. Dark meat White meat . Skin Depot fat percent 1 22. 0 32. 1 1 5 0. 5 2 21. 8 55. 0 2. 3 1 l 3 23. 2 50. 3 2 2 0. 6 4 l7. 0 51. 0 1 8 1 4 5 20. 5 40. 0 — - Average 21.0 48.0 2 0 0.9 1Phospholipids plus neutral lipids equal 100 percent. 2 . . . Each figure represents at least two determinations. 45 APPENDIX E (b) Figure 1. (a) Series of columns with control valve. (b) Valve detail. "minimItijjjjit