Inhibition of sperm motility and agglutination of sperm cells by free fatty acids in whole semen*

Vol. 45, No.2, February 1986 Printed in U.SA.

FERTILITY AND STERILITY Copyright 0 1986 The American Fertility Society

Inhibition of sperm motility and agglutination of sperm cells by free fatty acids in whole semen*

Israel Siegel, Ph.D. t Alan B. Dudkiewicz, Ph.D. Jan Friberg, M.D., Ph.D. Miguel Suarez, M.D. Norbert Gleicher, M.D. Division of Reproductive Immunology, Department of Obstetrics and Gynecology, Mount Sinai Hospital Medical Center and Rush Medical College, Chicago, Illinois

The effects of a serial dilution of linoleic acid on human spermatozoa in whole semen was tested on 21 semen samples obtained from 11 normal volunteers. The minimal concentration of linoleic acid required to stop the movement of at least 75% of the moving sperm ranged from 1 to> 100 mg/dl. Fifteen of21 (71%) of the semen samples were inhibited by added free fatty acids (FFA) concentrations that were less than or close to the physiologic concentration ranges of FFA in blood plasma (1 to 30 mg/dLJ. The immobilized sperm often formed aggregates similar to those formed by the action of autoantibodies against sperm cells. Preliminary studies conducted on a variety of other FFA have indicated that oleic acid (18/1) was less toxic than linoleic acid (18/2) and that linolenic acid (18/3) was more toxic than linoleic acid. The saturated FFA palmitic acid (16/0) and stearic acid (18/0) at concentrations up to 100 mg/dl showed little or no toxicity to sperm cells. It is suggested that FFA toxicity be included among physiologic factors that affect the motility and spontaneous aggregation of sperm cells. Fertil SteriI45:273, 1986

Free fatty acids (FFA) are highly toxic lipid molecules. Under conditions in which their toxic properties are not sufficiently neutralized, they induce toxic changes in red cells, neutrophils, platelets, fibroblasts, heart cells, and brain cells. 1 Our recent studies on the effects of parenteral fat emulsions on the immune adherence phenomenon 2 indicated that FFA liberated from the fat

Received December 3, 1984; revised and accepted November 1, 1985. *Supported by the Foundation for Reproductive Medicine, Inc., Chicago, Illinois. tReprint requests: Israel Siegel, Ph.D., Director, Reproductive Immunology Laboratory, Mount Sinai Hospital Medical Center, California Avenue at 15th Street, Chicago, Illinois 60608. Vol. 45, No.2, February 1986

emulsions induce toxic morphologic changes on red cells both in vitro and in vivo. 2 Additional studies in our laboratories have demonstrated that the nonsaturated FF A are toxic not only to red cells but also to tumor cells. 3 We then tested the possible cytotoxic effects of FF A on spermatozoa. In response to the addition of physiologic or lower-than-physiologic concentrations of FFA to semen, a dramatic inhibition of sperm movement often occurred. 4 This article describes the conditions under which FFA are toxic to spermatozoa in whole semen. MATERIALS AND METHODS

We obtained 21 semen samples from 11 normal human volunteers with proven fertility. The seSiegel et al. Toxicity of free fatty acid to sperm cells

273

men samples contained at least 60 x 10° sperml ml and < 30% abnormal forms and had > 50% motility. All semens were stored at room temperature and used during the day of ejaculation. SERUM

Human blood was obtained from the arm vein of normal volunteers. The blood was allowed to clot, and then the red cells and serum were separated. The serum was stored at - 70°C until use. FATTY ACIDS

Oleic (18/1), linoleic (18/2), linolenic (1813), stearic (18/0), and palmitic (1610) acids, all approximately 99% pure, were obtained from Sigma Chemical Company, St. Louis, MO. SALT SOLUTION

All cells were washed, and all fatty acids were diluted in a modified Hanks' balanced salt solution,5 except when otherwise stated. The salt solution contained 1.28 x 10- 1 M NaCI, 1 x 10- 2 M KCI, 2.87 x 10- 3 M K 2 HP0 4 , 8.8 x 1O-~ M KH2 P04 , and 1.7 x 10- 4 M HC!. FREE FATTY ACID TOXICITY ASSAYS

Assays of non saturated FFA toxicity on sperm were conducted in autologous semen as follows. Semen was distributed in 12 x 75-mm disposable glass tubes in O.I-ml portions, except for one tube (tube 1), which contained 0.2 ml of semen. Two microliters of the nonsaturated fatty acids were transferred to the tube .containing 0.2 ml of semen. The mixture was shaken for several seconds in a mechanical shaker to yield a semen fatty acid emulsion containing 1000 mgldl of the FFA. Then 1f1O ml of this mixture was transferred to a tube that contained 0.1 ml of semen, yielding a mixture containing 500 mgldl of FFA. The mixture was again shaken as described above. Such serial dilutions were continued until the fatty acids were diluted to the desired concentrations. Alternatively, all dilutions were made with half of the components listed above in 1.5-ml polypropylene Eppendorf Flex Micro Test Tubes (Brinkman Instruments Company, Westbury, NY). Because saturated fatty acids are solid at room temperature, they were dissolved at a 10% concentration in hot absolute alcohol. Then 2 J.LI of the 10% FFA was transferred to a tube containing 0.2 ml of semen, yielding an FFA concentration of 274

Siegel et al. Toxicity of free fatty acid to sperm cells

103 . The saturated FFA semen mixtures were then serially diluted exactly as described for the nonsaturated FF A. Controls included semenalcohol mixtures with alcohol concentrations similar to those in the semen and alcohol-dissolved FFA mixtures. In experiments comparing the toxicity of saturated and unsaturated FFA, both the saturated and unsaturated FFA were dissolved in hot alcohol. In several comparative experiments, the FFA were diluted in seminal plasma instead of semen, as follows. Semen samples were centrifuged at 500 x g for 30 minutes and the supernatant seminal plasma separated from the sedimented sperm cells. The FFA were then serially diluted in the seminal plasma according to the procedures described above for the semen. A portion of the sperm sediment was then added to the FFA-seminal plasma mixtures in quantities that would yield a sperm count similar to that in the untreated' semen. All assays included control mixtures, which contained sperm cells but no FF A. Pipette tips were changed for each serial transfer of the fatty acid mixtures. The mixtures were then transferred to a 37°C incubator flushed with 5% CO2 and incubated for 60 minutes. Then 25 J.LI of the mixtures was transferred to a microscopic slide and observed under the phase microscope. We counted,100 sperm cells at random to determine the percentage of moving sperm. The fact that serial dilutions of the FFA were made in semen introduces a potential source of error, because sperm are transferred together with the FFA during the serial dilution procedures. This could cause a maximum of 50% inhibition of the moving sperm in a serially diluted semen-FFA mixture preceded by a FFA-semen mixture in which all the sperm were nonmotile. To compensate for this and for counting errors, we considered only inhibitions that stopped> 75% of the moving sperm as statistically significant differences between individual control and FFAtreated mixtures (P < 0.001, chi-square analysis). Duplicate serially diluted mixtures indicated a twofold to fourfold error in the minimal toxic concentration of the FFA. ASCORBIC ACID EFFECTS

L-ascorbic acid (Sigma Chemical Company) was dissolved in the salt solution at a 1% concentration. The pH of the ascorbic acid salt solution was then adjusted to 7.2 with additions of IN Fertility and Sterility

Table 1. Minimal Concentrations of Added Linoleic Acids that Immobilized> 75% of the Sperm in Semen Donor

% Moving sperm FFA treated Controls

Minimal FFA required mg/dl

1 2 3 4 5 6 6 6 6 7 8 6 9 9 10 7 9 11 6 6 9

0 14 3 4 0 17 0 0 10 14 13 11 6 4 13 6 25 a 3 17 3 8

87 56 88 86 62 91 77 91 97 81 71 67 88 97 90 87 79 95 91 19 81

3.8 100 7.5 15 15 30 30 60 60 15 30 30 60 50 12 6 100 100 25 25 1

a·Lower value not available.

NaOH solution. Assays to test the effects of ascorbic acid were conducted as follows. Semen samples were distributed in 0.09-ml portions in tubes, except for one tube that received 0.18 ml of semen. Then 10 fl.l of 1 % ascorbic acid was added to the 0.09-ml portions of semen and 20 fl.l of 1% ascorbic acid to the 0.18-ml portion of semen. Thus, each mixture contained 5.7 mM of ascorbic acid. Control mixtures contained similar quantities of semen, except that salt solution, instead of ascorbic acid, was added. Linoleic acid was serially diluted in the semenascorbic acid or semen-salt solution mixtures exactly as described above for the pure semen mixtures. The mixtures were then observed and assayed as described for assay procedures in whole semen.

RESULTS INHIBITION OF SPERM MOTILITY

The effects oflinoleic acid on the sperm motility of 21 different semen specimens are summarized in Table 1. The minimal concentration of linoleic acid required to stop the movement of> 75% of the moving sperm varied in different semen samples, ranging from 1 mg/dl to > 100 mg/dl. Fifteen Vol. 45, No.2, February 1986

of 21 semen samples (71 %) were inhibited by FF A concentrations of ~ 30 mg/dl. The concentrations of FFA required to abolish sperm movement completely in the semen samples were available for 16 semen samples. The concentrations were the same as those required to inhibit 75% of the moving sperm in 4 of 16 (25%), 2 times larger in 6 of 16 (38%), 4 times larger in 4 of 16 (25%), 8 times larger in 1 of 16 (6%), and 100 times larger in 1 of 16 (6%) semen samples.

AGGLUTINATION OF SPERM CELLS

In addition to effects on sperm motility, the FFA caused agglutination of the sperm cells. In general, the nonmotile sperm usually formed aggregates and the motile sperm did not agglutinate. This resulted in a remarkable similarity between the proportion of motile and nonmotile sperm and the proportion of agglutinated and nonagglutinated sperm (Table 2) when counted in the same microscopic field.

EFFECTS OF INCUBATION PERIODS

In preliminary experiments, we compared the effects of different incubation periods on the FF A toxicity titers. Incubation ofsemen-FFA mixtures for 2 to 3 hours did not significantly increase the FFA toxicity oyer that observed after 1 hour of incubation. However, prolonged incubation of the mixtures often increased the FFA toxicity titers by about 2 times. Table 2. Effect of Linoleic Acid on Sperm Agglutination and Motility Linoleic acid concentration

%

Agglutinateda

%

Nonmotilea

mg/dl

1000 500 250 125 62 31 15 7.5 3.75 1.9 1.0 2.5 0

100 100 100 100 100 94 86 68 30 38 7 7 27

100 100 100 100 100 100 86 69 30 38 7 31 10

aAgglutinated sperm and nonmotile sperm were counted in the same microscopic fields. Linoleic acid was added to whole semen. Siegel et aI.

Toxicity of free fatty acid to sperm cells

275

Table 3. Reversibility of Toxic Effects of Linoleic Acid on Sperm Motility Linoleic· acid concentration

% of motile sperm Before removal After removal ofFFAa ofFFA b

mgldl

o o o

100 50

25 12.6 6 None

o

sperm movement than linoleic acid and that linolenic acid (18/3) was more inhibitory than linoleic acid. This is illustrated in Tables 5 and 6. In contrast to the unsaturated FFA, there were little or no inhibitory effects on sperm movement by the saturated 16- and 18-carbon FFA (Table 7).

33 62

4

80

55

87

86

67

aSpermatozoa were exposed to the FFA in pure autologous semen and incubated in a 37°C incubator for 60 minutes. bMixtures were sedimented to remove the semen~fatty acid supernatant. The spermatozoa were resuspended in physiologic salt solution containing 10% human serum.

DILUTION OF FREE FATTY ACIDS IN SEMINAL PLASMA

In four semen samples, linoleic acid was diluted in seminal plasma as well as in semen. There were no significant differences in the toxicities of the FFA between mixtures diluted in. semen or seminal plasma. REVERSIBILITY OF SPERM INHIBITION

To test the reversibility of the FFA effects on sperm movement, we sedimented the FFA-immobilized sperm cells to remove FFA and resuspended the sperm cells in physiologic salt solution containing 10% human serum. Typical results are illustrated in Table 3. The inhibition of sperm movement was irreversible or only partially reversible when sperm were exposed to toxic FFA concentrations at least 2 to 4 times larger than the minimal toxic concentrations of the FFA. EFFECT OF ASCORBIC ACID

To test the effects of ascorbic acid (an antiperoxidant) on the toxicity of FFA on sperm cells, we exposed sperm cells of four different semen samples to serial dilutions of linoleic acid in the presence or absence of ascorbic acid. Typical results are summarized in Table 4. Ascorbic acid did not affect the toxic effects of linoleic acid.

DISCUSSION

FFA have been known to constitute the most. metabolically active 6 and toxic lipid components. 7 This study is the first demonstration of the toxic properties of physiologic FFA on sperm cells; The average FFA concentration in human serum, as reported by different investigators, is 0.4 to 0.8 mEqll (11.2 to 22.4 mg/dl, assuming a·molecular weight of the FFA of 280) (range, 0.3 to 1.2 mEq/l; 8.4 to 33.6 mg/dl).l Thus the concentration of added FFA required to inhibit the movements of most of the sperm samples in this study was within or less than the physiologic concentrations of FFA in the blood plasma. The methods we used in this study were designed to detect relatively large effects; such as complete cessation of movement of most of the sperm cells or agglutination of the sperm cells. Additional studies. are required to determine the effects of FFA, using more subtle FFA-induced changes on sperm cells. These may consist of changes in the rate of sperm movement or FFA-induced ultrastructural changes in the sperm cells, which are likely to occur at FF A concentrations below those reported in this study .. The significance of toxic properties of physiologic FFA on sperm cells and the fertile state is at present unknown. To answer this question, more information regarding the normal concentrations of FFA and factors that neutralize FFA toxicity in semen is needed. Table 4. Ascorbic Acid and FFA Toxicity Linoleic acid

mgldl

1000 TOXIC EFFECTS OF OTHER FREE FATTY ACIDS

500

In preliminary studies, we compared the toxicities of oleic acid (18/1) and linolenic acid. (18/3) to that of linoleic acid (18/2). Comparative results obtained from four different semen samplesindicated that oleic acid (18/1) was Jess inhibitory to

125

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Toxicity of free fatty acid to sperm cells

% Sperm motility No ascorbic Ascorbic acid (5.7mM) acid

250

o o o o

o o o o

32.5

10

10

16.25 8.1 4

74

69

97 94 100

88 95

o

72

Fertility and Sterility

Table 5. Effects of Linoleic Acid (18/2) and Linolenic Acid (18/3) on Sperm Motility in Vitro FFA concentration

% Sperm motility Linolenic acid Linoleic acid (18/2)

(18/3)

o

o o o

mg/dl

100 50 25 12.5 6.25 3

o

15 11

46 54

74 67

2 63 62

50

Only relatively few studies have been done to determine FFA concentrations in either semen or seminal plasma. Scott et al. 8 measured FFA in semen of a variety of species that did not include man. The concentration of FFA ranged from 0.1 mEq/1 (0.28 mg/dI) in rabbits to 0.21 mEqll (5.88 mg/dI) in rams. Brooks et al. 9 reported an average FFA concentration in epididymal plasma of rats of 0.958 ± 0.156 mEq/1 (26.8 ± 4.4 mg/dI). This was about 4 times higher than the corresponding FFA concentration in rat blood plasma (0.230 ± 0.057 mEq/l; 6.4 ± 1.6 mg/dI). Abdel et aLlo found a FFA concentration of 8.97 mg/dl (0.32 mEqll) in human semen. This is within the lower concentration ranges of FFA in human plasma. The major plasma component that neutralizes fatty acid toxicity is albumin. l Thus in the presence of albumin, FFA toxicity is a function of the FFA/albumin ratio. The ability of albumin to neutralize FF A toxicity in plasma efficiently seems to be limited to about two FFA per albumin molecule. l For example, an FFAlalbumin ratio of 2 to 3 inhibits chemotaxis of neutrophils by about 50%, 1 makes platelets more susceptible to aggregation,1 and induces toxic morphologic transformation of human red cells. 6 A ratio of 3 was demonstrated to depress the contractility of rat heart preparations. 1 A ratio of4 depresses phagocytosis and bactericidal abilities of neutrophilsl and displaces bound bilirubin from albumin molecules. l l A ratio of 5 changed enzymatic patterns in perfused heart muscle. 1 A ratio of7 to 8 causes heart abnormalities in patients with myocardial infarctions. 1 We could find no studies done to determine the FFA/albumin ratios in semen or seminal plasma. Several authors, however, reported that the albumin concentration in whole semen in men is about 1% to 2% of the albumin concentration in blood plasma. 12, 13 It can be calculated from these Vol. 45, No.2, February 1986

reports that the FF A/albumin ratio in semen is about 50, an extraordinary high ratio that requires confirmation. Additional. studies are also required to relate the FFA/albumin ratios to the susceptibility of sperm cells to added FF A .. We suggest that the relatively low concentrations of albumin in semen may account. in part for the susceptibility of sperm in semen to FFA. Our results indicate that different semen samples may vary in their susceptibility to the toxic effects of FFA. These variations exceeded the inherent errors in the procedures. The variations were evident not only in semen samples of different donors, but also in semen samples obtained at different times from the same donor (donor 9, Table 1). The factors that cause this variability are unknown and require further investigation. One possible factor may be the natural variation in the concentration of albumin in semen. For example, Hekman and Rumke 12 showed a threefold to fourfold difference in the albumin concentration between ejaculates· of different subjects, which greatly exceeds the natural variations evident in albumin concentrations of normal serum. Our studies indicate that the toxicities of FFA were dependent· not only onthe concentrations of FFA but also on the type of FFA in the reaction mixtures. The nonsaturated I8-carbon FF A were more toxic than the saturated 16- to 18-carbon FFA. This is consistent with the results of avariety of studies that have indicated that the nonsaturated FF A are generally more toxic to tumor cells3, 14-16 and bacteria l7 than saturated fatty acids. In recent studies in our laboratory, we have likewise demonstrated that the 16- to 18-carbon FF A were more toxic to mammalian red cells than the 16- to 18-carbon saturated FFA. 6 In addition, differences in toxicities to sperm cells were evident between different nonsaturated 18-carbon FFA. The fact that oleic acid (18/1) was less Table 6. Effects of Linoleic Acid (18/2) and Oleic Acid (18/1) on Sperm Motility in Vitro Fatty acid concentration

% Sperm motility Oleic acid

Linoleic acid (18/2)

(18/1)

rRg/dl

100

50 25 12.5 6 3 1.5

o

o o o 8.4 14.5 19.3 45 93.4

2 8.2

10 15.6 27.3 38 36

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Table 7. Effects of Stearic (18/0), Palmitic (16/0), and Linoleic Acid (18/2) on Sperm Motility in Vitro % Sperm motility

FFA concentration Stearic (18/0)

Palmitic (16/0)

Linoleic (1812)

mgldl

100 50 25 12.5 6.2 0

55 53 82 84 73 71

62 73 67 77 84

0 2 13 31 71

toxic to sperm cells than linoleic acid (18/2) and linolenic acid (18/3) is consistent with other comparative studies that have indicated that oleic acid (18/1) was the least toxic 18-carbon FF A to a variety of tumor cells. 14-16 Hamfler et al. 18 determined the percentages of different FFA in human semen. The percentage of linoleic acid was 6.15 ± 1.88, and that of oleic acid was 13.04 ± 1.94. Thus the two unsaturated FFAconstituted about 20% of the total FFA in semen. The percentage of linolenic acid (which was the most toxic of the 18-carbon FF A) and the absolute concentration of the FFA were not determined in Hamfler's study. The mechanism through which FF A are toxic to cells is still unknown. FF A are surface-active agents,19 but changes in surface tension alone cannot completely account for the toxicities of the FFA. 19 Unsaturated fatty acids are generally highly susceptible to peroxidation. Jones et al. 20 demonstrated that exogenously applied peroxides are powerfully spermicidal. Nissen and Kreyse1 21 found that poorly motile spermatozoa showed a relatively high rate of endogenous lipid peroxidation. We therefore considered the possibility that the toxic action of the linoleic acid occurred through the peroxides formed from linoleic acid. To test this hypothesis, we exposed the FFA sperm mixtures to ascorbic acid, a known antiperoxidant. If toxicity occurred through peroxidation of linoleic acid, it would be diminished by the presence of ascorbic acid in the mixture. Our results showed no decrease in FFA toxicity by the presence of ascorbic acid (Table 4). This suggests that the toxicity of linoleic acid to sperm cells does not occur through the peroxidation of linoleic acid. FFA levels in vivo have been known to increase to above their average value in response to a variety of common life conditions. For example, 278

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Toxicity of free fatty acid to sperm cells

plasma concentrations of FF A have been known to increase up to two or three times their average values in response to ingestion of a fatty meal,22 smoking,23 caffeine,24 and stress. 25 It is at present not known how these fluctuations of FFA levels affect FFA levels in the semen. Further research is therefore required to determine how such environmental factors affect the levels of FFA in semen and, thus, sperm motility. Agglutination of sperm cells and reduction in sperm motility have been considered the hallmarks of the toxic action of autoantibodies on sperm cells. Our studies indicate that such effects may be produced not only by antibodies, but also by changes in natural FFA concentrations. We therefore suggest that along with antibodies and other factors, FFA toxicity should be considered during clinical evaluations of spontaneous reductions of sperm motility and agglutinations of sperm cells.

REFERENCES 1. Spector AA, Fletcher JE: Transport of fatty acid. In Disturbance in Lipid and Lipoprotein Metabolism, Edited by JM Dietschy, AM Gotto, JA Ontko. Bethesda, American Physiological Society, 1978, p 22 2. Siegel I, Liu TL, Zaret P, Gleicher N: Parenteral fat emulsions and immune adherence. The effects of triglycerides on red cell and neutrophil immune adherence in vitro and in vivo. JAMA 251:1574, 1984 3. Siegel I, Gleicher N: The selective toxicities of nonsaturated fatty acids upon mammary ascites tumor cells. Am J Reprod Immunol (Abstr) 6:74, 1984 4. SiegeU, Dudkiewicz A, Friberg J, Gleicher N: The effects of free fatty acids on human sperm cells. Am J Reprod Immunol (Abstr) 6:59, 1984 5. Martin SP, Green R: Methods for the study of surviving leukocytes. In Methods in Medical Research, Vol 7, Edited by J Warren. Chicago, Year Book Medical Publisher, 1958, p 136 6. Siegel I, Gleicher N: Unpublished data 7. Kigoshi S, Ito R: High levels of free fatty acids in lymphoid cells with special reference to their cytotoxicity. Experientia 29:1408, 1975 8. Scott TW, White IG, Annison EF: Fatty acids in semen. Biochem J 78:740, 1961 9. Brooks DE, Hamilton DW, Mallek AH: Carnitine and glycerylphosphorylcholine in the reproductive tract ofthe male rat. J Reprod Fertil 36:141, 1974 10. Abdel Z, El-Haggar SH, Tawadrous GA, Hamada T, Shawky MA, Amin K: Seminal lipids as energy substrate for the spermatozoa. Andrologia 15:259, 1983 11. Kerner JA, Cassani C, Hurwitz R, Berde CB: Monitoring intravenous fat emulsion in neonates with the fatty acid! serum albumin molar.ratios. JPEN 5:51, 1981

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12. Hekman A, Rumke P: Seminal antigens and autoimmunity. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St. Louis, C. V. Mosby, 1976, p 245 13. Tauber PF, ZanE!veld LJD, Propping D, Schumacher GFB: Components of human split ejaculates. I. Spermatozoa, fructose, immunoglobulins, albumin, lactoferrin, transferrin and other plasma proteins. J Reprod Fertil 43:249, 1975 14. Ando K, Kato A, Kimura T, Suzuki S, Tamuro G, Arima K: Antitumor activity of fatty acids and their esters. I. Evaluation of antitumor activity of fatty acids. In Progress in Antimicrobial and Anticancer Chemotherapy, Proceedings of the Sixth International Congress of Chemotherapy. Baltimore, University Park Press, 1970; p 136 15. Katchman BJ, Zipf RE, Murphy PJ: The effects of fatty acids upon tumor cell respiration and transplantability. Clin Chem 9:530, 1963 16. Tolnai S, Morgan JF: Studies on the in vitro antitumor activity of fatty acids. V. Unsaturated acids. Can J Biochem Physiol 40:869, 1962 17. Nieman C: Influence oftrace amounts offatty acids on the growth of microorganisms. Bacteriol Rev 18: 147, 1954

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18. Hamfler HW, Nissen HP, Heinze I, Kreysel HW, Schirren C: Analyse der freien Fettsauren des Humanspermas unter diagnostischen Aspekten. Andrologia 10:498, 1978 19. Glassman HN: Surface active agents and their application in bacteriology. Bacteriol Rev 12:105, 1948 20. Jones R, Mann T, Sherins R: Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides, and protective action of seminal plasma. Fertil Steril 31:531, 1979 21. Nissen HP, Kreysel HW: Polyunsaturated fatty acids in relation to sperm motility. Andrologia 15:265, 1983 22. Castelli WP, Nickerson RJ, Newell JM, Rutstein DD: Serum NEFA following fat, carbohydrate and protein ingestion, and during fasting as related to intracellular lipid deposition. J Atheroscler Res 6:328, 1966 23. Kershbaum A, Bellet S: Cigarette smoking and blood lipids. JAMA 187:32, 1964 24. Patwardhan RV, Desmond PV, Johnson RF, Dunn GD, Robertson DH, Hoyumpa AM, Schenker S: Effects of caffeine on plasma free fatty acids, urinary catecholamines, and drug binding. Clin Pharmacol Ther 28:398, 1980 25. Bogdonoff MD, Estes EH Jr, Trout D: Acute effect of psychologic stimuli upon plasma non-esterified fatty acid level. Proc Soc Exp BioI Med 100:503, 1959

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