Phospholipid peroxidation in tumor promoter-exposed mouse skin.

We have investigated lipid peroxidation in the skin of CD1 mice following single or repeated topical applications of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). A substantial accumulation of hydroxyphospholipids, to levels 3-5 times control values, followed exposure to two or more TPA treatments (24-72 h intervals), whereas single applications were ineffective. Sodium borohydride reduction increased the yield of product by approximately 50%, suggesting the additional presence of phospholipid hydroperoxides in the oxidized lipids. Straight phase HPLC analysis of the constituent hydroxy fatty acids, followed by gas chromatography/mass spectrometry, revealed that oxidized derivatives of linoleic acid, including 9- and 13-hydroxyoctadecadienoic acids (9- and 13-HODE), were the primary products. Stereochemical analysis showed ratios of S to R stereoisomers of 1.3 for 13-HODE and 1.27 for 9-HODE, which implied that TPA-induced peroxidation was primarily due to free radical oxidation, although a partial contribution of enzyme (lipoxygenase) activity is possible. The TPA-induced peroxidation was greater in the epidermis than in the dermis. Pre-exposure of mouse skin to the anti-inflammatory agent fluocinolone acetonide, antioxidants and enzyme (phospholipase A2 and lipoxygenase) inhibitors lowered the peroxidation response to subsequent exposure to TPA. Phospholipid peroxidation products may be useful markers of oxygen radical production in TPA-exposed mouse skin with possible relevance to tumor promotion.

Induction of manganese superoxide dismutase by glucocorticoids in glomerular cells.

Our previous in vivo study demonstrated that methylprednisolone (MP) activates glomerular antioxidant enzymes and attenuates glomerular oxidant injuries, including those in experimental nephrosis. The present study investigates the cellular mechanism of the MP-induced activation of antioxidant enzymes and their contribution to the attenuation of cellular oxidant toxicity. When bovine glomerular endothelial cells (GECs) were treated with 10 microM MP, cellular manganese superoxide dismutase (Mn-SOD, 3.95 +/- 0.33 mu/mg protein, M +/- SE) and catalase (1.64 +/- 0.06 k/mg protein) activities were significantly (P < 0.05) elevated above control GECs (2.23 +/- 0.43 mu/mg protein and 1.06 +/- 0.09 k/mg protein, respectively). When GECs pretreated with MP (10 microM 24 hrs) were exposed to xanthine (0.1 mM)+xanthine oxidase (5 mU/ml) for four hours, levels of specific membrane lipid peroxidation products, that is, phosphatidylcholine- and phosphatidylethanolamine-hydroperoxides, remained at levels 10 to 25% of those measured in non-MP-treated (xanthine/xanthine oxidase-exposed) control cells. Moreover, the degree of cell damage following exposure to the superoxide generating system, assessed by 51Cr release, was significantly attenuated in MP-treated cells (approximately 50% of MP-non-treated controls, N = 6). Thus, MP-treated GECs with elevated antioxidant enzyme activities by MP were more resistant to the toxic effect of reactive oxygen metabolites. The mechanism of antioxidant enzyme induction by MP was studied for Mn-SOD. MP was shown to enhance Mn-SOD mRNA in bovine GECs and rat glomerular mesangial cells (GMCs) in dose-dependent manners.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced activity of antioxidant enzymes underlies contrast media-induced renal injury in volume depletion.

Oxidant-mediated renal injury has been suggested as an important mechanism of acute renal failure induced by contrast media. Since volume depletion has been recognized as a predisposing factor for contrast media nephropathy, the present study was designed to characterize host-defense mechanisms against oxidant-mediated renal injury during volume depletion. Antioxidant enzyme activities in renal cortex were compared between acutely water deprived (WD, 72 hours) and non-WD rats. WD rats had reduced activities of catalase and superoxide dismutase activities (on average, 48% and 60% of values in non-WD, respectively). In separate groups of WD rats, saline or one of three different contrast media, namely diatrizoate meglumine/diatrizoate sodium (DTZ), ioxaglate meglumine/ioxaglate sodium (IXG), and iohexol (IHX) was injected. Both GFR and renal plasma flow rate, measured 24 hours later, was some 50% less in DTZ-injected than saline-injected WD rats. WD rats treated with IXG and IHX had similar GFR to saline-treated rats. In DTZ-treated WD rats, specific products of membrane lipid peroxidation, phosphatidylcholine and phosphatidylethanolamine hydroperoxide, determined by chemiluminescent HPLC, were more than two-fold higher than saline, IXG, or IHX-treated WD rats. DTZ did not induce renal dysfunction and enhance lipid peroxidation in non-WD rats. Therefore, DTZ appeared to induce oxidant-mediated injury only in WD rats. When WD rats were pretreated with polyethylene glycol-coupled catalase (1.4 mg x 2 days), renal cortical catalase activity remained at a level similar to that of non-WD rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of aldehydic lipid peroxidation products by TLC/densitometry.

We have explored the use of thin-layer chromatography (TLC)/densitometry in both the reflectance and fluorescence mode for quantitation of specific products of lipid peroxidation. Aldehydic peroxidation products were generated by exposure of arachidonic acid to iron and ascorbic acid for 24 hr. Several methods for the quantitative analysis of peroxidation products by TLC/densitometry were compared using two different aldehyde-specific derivatizing reagents, namely dinitrophenylhydrazine (DNPH) and cyclohexanedione (CHD). DNPH hydrazones of the arachidonic acid-peroxidation products, upon TLC separation on silica gel, revealed prominent alkanal and hydroxyalkenal bands. Reverse phase high performance liquid chromatography confirmed that the primary alkanal component was hexanal, while the primary hydroxyalkenal was 4-hydroxynoneal. Semiquantitative methods for the direct analysis of these products by TLC/densitometry were worked out based on the use of external hydrazone standards. TLC/densitometry (fluorescence mode) was used to measure CHD adducts of aldehydes by forming the derivatives in the presence of decanal (used as an internal standard) and separating the derivatives by reverse phase TLC. Hexanal-CHD was detectable upon application of 0.5 nanomoles while 4-hydroxynoneal showed a lower response and was detectable with 10 nanomoles. Using appropriate response factors, hexanal and 4-hydroxynonenal were measured in the aldehyde sample from arachidonic acid and results were similar to those obtained by the DNPH method. Similar approaches were used to analyze the peroxidation products of docosahexaenoic acid (24-hr exposure) and of rat liver microsomes exposed to iron for 30 min. The DHA peroxidation products contained extremely low levels of alkanals, while polar aldehydes and hydroxyalkenals were prominent. Formation of alkanals, osa-zones, hydroxyalkenals and phospholipid aldehydes from iron-expoded microsomes was also demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic changes in phospholipid hydroperoxide levels during renal ischemia/reperfusion.

The involvement of lipid peroxidation in renal ischemia/reperfusion was explored by measuring changes in the cortical content of specific primary lipid hydroperoxides (using chemluminescent detection with HPLC) following ischemia and reperfusion and by correlating the changes in hydroperoxide content with measurements of renal blood flow. Phosphatidylcholine and phosphatidylethanolamine hydroperoxide concentrations were significantly lowered during 30 or 60 min of ischemia (to levels less than 50% of control at 60 min). Following 30 min of renal ischemia, reperfusion resulted in a rebound of phospholipid hydroperoxide tissue content to levels higher than controls. Increased phospholipid hydroperoxide formation was not, however, observed in response to reperfusion following long-term (60 min) ischemia. In separate animals it was demonstrated that following 30 min ischemia and reperfusion, renal blood flow recovers to about 65% of control in 1 h. In contrast, following 60 min ischemia and reperfusion, the renal blood flow remains more highly impaired (less than 25% recovery for periods up to 24 h). These results imply that phospholipid hydroperoxides are produced and accumulate in the kidneys under normal aerobic conditions and that lipid peroxidative activity increases during renal ischemia/reperfusion to an extent dependent on the degree of local blood perfusion.

Identification of hydroxyalkenals formed from omega-3 fatty acids.

The highly toxic lipid peroxidation product, 4-hydroxynonenal, is formed from the decomposition of hydroperoxides of omega-6 fatty acids. In this study the analogous hydroxyalkenals formed from the decomposition of hydroperoxides of omega-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) were isolated and identified using TLC densitometry, HPLC and GC/Mass Spectrometry. The major hydroxyalkenal formed from both fatty acids was a diene analog of 4-hydroxynonenal, 4-hydroxynona(2,6)dienal, while 4-hydroxyhexanal was a minor product. Measurement of specific omega-3 lipid peroxidation products may be important in studies using dietary fish oil.

Effects of aurothioglucose on iron-induced rat liver microsomal lipid peroxidation.

Aurothioglucose (ATG), an inhibitor of selenium-dependent glutathione peroxidase activity, at a concentration of 100 microM, strongly increases lipid peroxidation of rat liver microsomes exposed to either ferrous ion (10 microM) or the combination of ferric ion (10 microM) and ascorbic acid (500 microM), in the presence of reduced glutathione (GSH, 800 microM). This effect was not achieved using heat-inactivated microsomes and was dependent on the presence of GSH. ATG did not affect the lag period associated with ascorbic acid/ferric ion-induced microsomal lipid peroxidation (previously attributed to an undefined GSH-dependent microsomal agent), but did increase the rate of peroxidation subsequent to the lag period. The potent GSH-dependent inhibition of microsomal lipid peroxidation by cytosol (10% of total volume) was completely reversed by ATG (100 microM). ATG similarly reversed an inhibition of phosphatidylcholine hydroperoxide-dependent liposomal peroxidation that has been attributed to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme distinct from the classical glutathione that cannot utilize intact phospholipids. ATG inhibited, in addition to the classical selenium-dependent glutathione peroxidase, both cytosolic and microsomal (basal and N-ethyl maleimide-stimulated) glutathione S-transferase activities with greater than 80% inhibition achieved at 100 microM ATG. ATG, at concentrations up to 250 microM, did not inhibit PHGPX activity measured by the coupled-enzyme method in the presence of Triton X-100 (0.1%).(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione redox pathway and reperfusion injury. Effect of N-acetylcysteine on infarct size and ventricular function.

Glutathione peroxidase is an important enzyme in the degradative cascade of reactive oxygen free radicals. N-Acetylcysteine (NAC) is a low molecular weight compound that has been used clinically to replenish glutathione. To assess the role of the glutathione redox pathway on reperfusion injury, 23 animals underwent 90 minutes of proximal left anterior descending coronary artery occlusion followed by 24 hours of reperfusion with the administration of NAC (n = 11) or saline (n = 12) beginning 30 minutes into occlusion and continuing for 3 hours after reperfusion. Regional ventricular function was measured with contrast ventriculography, and regional myocardial blood flow was determined with microspheres. At 24 hours, the area at risk was defined in vivo with Monastral Blue, and the area of necrosis was defined by incubation in triphenyltetrazolium. Biopsies were taken from the ischemic and nonischemic zones to determine levels of total glutathione, superoxide dismutase and glutathione peroxidase activity, and reactivity to thiobarbituric acid, an index of lipid peroxidation. The rate-pressure product and myocardial blood flow were similar in the two groups throughout the study. No significant differences were noted in infarct size expressed as a percentage of the area at risk (28.6 +/- 5.3% vs. 36.6 +/- 6.0%) and of the total left ventricle (14.4 +/- 3.2% vs. 16.5 +/- 3.1%), and no differences were noted between the two groups on examination of the ischemic subendocardium by light and electron microscopy. Both groups exhibited similar degrees of dyskinesis during occlusion; however, treated animals showed significant improvement in regional radial shortening at 3 hours (3.4 +/- 2.4% vs. -2.4 +/- 2.1%, p less than 0.02) and 24 hours (9.2 +/- 2.2% vs. -2.5 +/- 6.3%, p less than 0.001) after reperfusion. No differences were present in total glutathione, thiobarbituric acid reactivity, or superoxide dismutase and glutathione peroxidase activity in the ischemic zones of the two groups. This study suggests that N-acetylcysteine treatment before reperfusion may reduce myocardial stunning but does not limit myocyte death after reperfusion.

Promotion of iron-induced rat liver microsomal lipid peroxidation by copper.

Although copper has been demonstrated to promote lipid peroxidation in a number of systems, the mechanisms involved have not been fully defined. In this study, the role of copper in modifying lipid peroxidation has been explored in rat hepatic microsomes. In an in vitro system containing reduced glutathione (GSH, 200 microM) and Tris buffer, pH 7.4, cupric sulfate (1-50 microM) potentiated lipid peroxidation induced by ferrous sulfate (10 microM) but was unable to elicit peroxidation in the absence of iron. Higher levels of cupric sulfate (100 microM or greater) were inhibitory. The nature as well as the extent of the peroxidative response of microsomes to cupric sulfate were dependent on glutathione levels in addition to those of iron. Cupric sulfate (100 microM) strongly potentiated ferrous ion-induced lipid peroxidation in the presence of 400-800 microM GSH, while it inhibited peroxidation at lower levels of GSH (0-200 microM) and did not affect ferrous ion-induced peroxidation with glutathione levels of 3-10 mM. The potentiating effect of copper on ferrous ion-induced lipid peroxidation was further explored by investigating: (1) potential GSH-mediated reduction of cupric ions; (2) potential copper/GSH-mediated reduction of ferric ions (formed by oxidation during incubation); and (3) possible promotion of propagation reactions by copper/GSH. Our results indicate that cupric ions are reduced by GSH and thus are converted from an inhibitor to an enhancer of iron-induced lipid peroxidation. Cuprous ions appear to potentiate lipid peroxidation by reduction of ferric ions, rather than by promoting propagation reactions. Iron (in a specific Fe+2/Fe+3 ratio) is then an effective promoter of initiation reactions.

The role of phospholipase A activity in rat liver microsomal lipid peroxidation.

The involvement of phospholipase(s) A in lipid peroxidation of rat liver microsomes was investigated by: (a) determining the effects of phospholipase A inhibitors (p-bromophenylacyl bromide, chlorpromazine, mepacrine) on the accumulation of thiobarbituric acid reactivity or on levels of oxidized phospholipids in response to selected oxidative stimuli and (b) measurement of phospholipase A activities in response to these agents. Lipid peroxidation in response to various peroxidation systems was inhibited completely by exposure of microsomes to p-bromophenylacyl bromide (250 microM). The effectiveness of p-bromophenylacyl bromide was dependent on the presence of glutathione (200 microM) in preincubation mixtures. Chlorpromazine (100 microM) and mepacrine (100 microM) also effectively inhibited peroxidation, and their potency was independent of glutathione. The accumulation of oxidized phospholipids in response to the potent peroxidation stimulus alloxan/ferrous ion was similarly inhibited by p-bromophenylacyl bromide, although the level of oxidized phospholipid in response to the initiator ADP/ferrous ion was not affected. Microsomal phospholipase A1 activity, assessed using a liposomal substrate, was substantially enhanced by promoters of lipid peroxidation. Phospholipase A2 activity was not detected using a liposomal substrate but was evident using radiolabeled microsomes as endogenous substrate and was enhanced by oxidative stimuli. We conclude that phospholipase A activity may play an integral role in the microsomal lipid peroxidation mechanism. Based on this study, we hypothesize a role for phospholipases in facilitating propagation reactions.

Modulation of neutrophil oxidative responses to soluble stimuli by platelet-activating factor.

The role of platelet activating factor (PAF) as a regulator of human neutrophil superoxide (O2-) generation in response to soluble and particulate stimuli was examined. At concentrations greater than 10(-7) mol/L, PAF alone induced a brief burst of O2- production. When cells were exposed to PAF and either the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP 10(-7) mol/L) or the tumor promoter phorbol myristate acetate (PMA 10 ng/mL), a marked synergistic augmentation of O2- release was noted when compared to control cells stimulated with FMLP or PMA alone. Mean percentage of enhancement by 10(-5) mol/L of PAF was 297% +/- 35% (n = 9) of control responses to FMLP and 185% +/- 16% (n = 3) of control responses to PMA. Consistent enhancement occurred with PAF concentrations of as low as 10(-9) mol/L. Enhancement could be demonstrated when neutrophils were exposed to PAF either at the same time as, or up to 60 minutes prior to, the second stimulus, and was neither reversed by removal of PAF from the medium prior to addition of FMLP or PMA nor dependent on the presence of extracellular divalent cations. Continuous recordings revealed that the enhancement was due to an increased maximal rate of O2- production. In contrast, PAF concentrations up to 10(-5) mol/L had only a minimal effect on the response to neutrophils to opsonized zymosan. Analysis of the enhancing properties of lipids structurally related to PAF revealed that the critical moiety was the saturated fatty acid at position 1. These results indicate the presence of a PAF-mediated positive feedback loop whereby the oxidative burst induced by some soluble stimuli is augmented. Modulation of neutrophil O2- production by PAF may serve to amplify neutrophil oxidative responses at sites of inflammation.

Investigations of the biological activity of leukotriene A4 in human polymorphonuclear leukocytes.

An investigation was undertaken to compare the responses of human neutrophils to the epoxide leukotriene A4 with those elicited by its stable product leukotriene B4 under identical IN VITRO conditions. LTA4 evokes neutrophil responses similar in nature to those induced by LTB4 but at much higher concentrations. Evidence suggests that LTA4 is important primarily for its role as an intermediate rather than for inherent activity.

Differential effects of lipoxygenase products on FMLP and LTB4 evoked neutrophil aggregation.

There is evidence that the endogenous biosynthesis of LTB4 is involved in the aggregation of human neutrophils induced by the chemotactic peptide f-met-leu-phe (FMLP). If LTB4 mediates this aggregatory response, then agents which desensitize neutrophils to LTB4 should inhibit the cellular response to FMLP. Since many lipoxygenase products modulate other neutrophil responses to LTB4 and FMLP, we have investigated the effects of lipoxygenase products on LTB4- and FMLP-initiated aggregation. Prior exposure to low concentrations of LTB4 (0.5-10 nM) inhibited subsequent aggregation to the same agent (50 nM), but it did not influence the response to FMLP (10(-7) M). Relatively high concentrations of 5-HETE (5-50 microM) inhibited aggregation initiated by either stimulus. Although the hydroperoxy derivative 5-HPETE also inhibited the response to LTB4, in the relatively narrow concentration range of 1-4 microM it stimulated FMLP-induced aggregation. This latter effect was confirmed using 12 cell preparations from six separate donors; it (the activity of 5-HPETE) was not mimicked by other 5-lipoxygenase products, including LTB4, nor the dihydroperoxide 8,15-DiHPETE. Our results indicate that neutrophil aggregation in response to LTB4 or FMLP can be selectively potentiated or inhibited. On the basis of these data we conclude that the endogenous synthesis of LTB4 is not directly involved in the neutrophil aggregatory response to FMLP, although the hydroperoxy intermediate 5-HPETE may act to enhance the cellular response.

Stimulation by lipoxygenase products of superoxide anion production in FMLP-treated neutrophils.

Our recent observation that leukotriene B4 (10(-9)M) is a potent enhancer of FMLP-initiated neutrophil superoxide anion formation prompted an evaluation of the ability of other lipoxygenase products and related compounds to modulate this response. The results indicate that FMLP-evoked O-2 may be enhanced by 10(-8)-10(-7)M levels of a number of lipids, in addition to LTB4, including 5-HPETE, 5-HETE, 5,15-DiHPETE and by higher levels of other 15-series lipoxygenase products and arachidonic acid. It is of interest that the relative potency of these agents in potentiating the superoxide response to FMLP approximately parallels their reported ability to induce chemotactic activity in leukocytes.

Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4.

Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.

Hydrolysis of sarcolemma by lysosomal lipases and inhibition by chlorpromazine.

The susceptibility of the lipids of canine cardiac sarcolemma to attack by soluble lysosomal lipases was studied to simulate in vitro the lipolytic injury that occurs during ischemia. The sarcolemmal fraction was incubated at 37 degrees C with the soluble portion of rat hepatic lysosomes (the lysosol) under conditions (pH 5.0, 5 mM ethylenediaminetetraacetic acid) appropriate for the activity of the major lysosomal lipases. Incubation of sarcolemma with lysosol resulted in a 78% lipolysis of sarcolemmal triacylglycerols, a lesser degradation of glycerophospholipids, and a parallel production of free fatty acids and lysophospholipids. The hydrolysis of sphingomyelin was negligible but was greatly stimulated (75%) by the addition of Triton X-100 (1 mg). Endogenous lipolytic activities of the sarcolemma did not contribute significantly to the observed lipid hydrolysis either in the presence or absence of detergent. The lipolysis of sarcolemmal triacylglycerols, glycerophospholipids, and sphingomyelin (Triton X-100 stimulated) were inhibited by varying concentrations of chlorpromazine. Thus cardiac sarcolemma is susceptible to hydrolysis of lysosomal lipases, and chlorpromazine inhibits this potentially injurious process.

Endogenous lipolytic activities during autolysis of highly enriched hepatic lysosomes.

High enriched (50- to 70-fold) fractions of "native" lysosomes were isolated using continuous flow electrophoresis from livers of rats which had not been pretreated with Triton WR-1339. Incubation of lysosomes for 30 min at pH 5.0 in the presence of 5 mM EDTA resulted in a dramatic loss in the content of fatty acids bound to triacylglycerols (137 down to 10 mumol/mg protein) and to phospholipids and an elevation in the level of unesterified fatty acid. Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin concentrations decreased whereas those of lysophosphatidylethanolamine (0.8 up to 8.5% of total lipid-P) and lysophosphatidylcholine (1.9 up to 16.7%) rose in a manner parallel to their respective, fully acylated lipids. Other phospholipids, including phosphatidylinositol, did not change in concentration during incubation. These results indicate that lysosomal phospholipase A, sphingomyelin and triacylglycerol lipase are activated by incubation at acid pH, enabling them to hydrolyze endogenous lysosomal lipids. However, lysosomal phosphatidylinositol-directed phospholipase C is apparently unable to interact with phosphatidylinositol of the lysosomal membrane.

The metabolism of polyunsaturated fatty acids in rat Sertoli and germinal cells.

The metabolism of [1-14C] linoleic, [1-14C] arachidonic and [3-14C] docosa-4,7,10,13,16-pentaenoic acids was investigated after intratesticular injection of the labeled compounds and isolation of rat Sertoli and germinal cell. Following injection of either 14C-linoleate or 14C-arachidonate, the specific activity (sp act) of docosa-4,7,10,13,16-pentaenoic acid of Sertoli cells was greater than that of the germinal cells. The data suggest that the Sertoli cells are more active in the biosynthesis of the 22-carbon pentaene than the germinal cells. Differences between these 2 cell types were also noted in the distribution of the incorporated 14C among the various lipid classes. Following intra-testicular injection of 14C-docosapentaenoic acid, a greater proportion of the recovered 14C in Sertoli cells than in germinal cells was present in 20-carton fatty acids, suggesting a greater activity in Sertoli cells in the metabolism of the pentaene. The major portion of the recovered 14C in both cell types was present in triacylglycerols during early time periods and in phospholipids after 24 hr. The possibility of transfer of biosynthesized docosapentaenoic acid from Sertoli to germinal cells is discussed.

A comparative study of the lipid composition of isolated rat Sertoli and germinal cells.

The lipid composition of enriched preparations of Sertoli cells and of germinal cells, isolated from the testes of mature rats, has been investigated. Sertoli cells contained a much lower content of phospholipids (in particular, much less phosphatidylcholine and phosphatidylethanolamine) and a higher content of triacylglycerols than did germinal cells. In addition, the Sertoli cells had a higher ratio of esterified to unesterified cholesterol than did germinal cells. Total lipids of Sertoli cells contained considerably lower levels of palmitic and docosa-4,7,10,13,16-pentaenoic acids and higher levels of stearic and oleic acid than did the total lipids of germinal cells. The major phospholipid classes and the triacylglycerols of Sertoli cells similarly contained less palmitic and docosa-4,7,10,13,16-pentaenoic acids, more stearic and oleic acids and also more arachidonic acid than did the corresponding lipid classes of the germinal cells. Minor differences between cell types were also noted for the content of palmitoleic, linoleic, docosa-7,10,13,16-tetraenoic, docosa-4,7,10,13,16,19-hexaenoic and tetracosa-9,12,15,18-tetraenoic acids.

The lipid composition of isolated rat spermatids and spermatocytes.

The lipids composition of enriched fractions of spermatids and spermatocytes, isolated from rat testicular tissue, has been investigated. More than 20% of the total fatty acids of spermatids but only 10% of those of spermatocytes, isolated from testes of mature rats, was 4,7,10,13,16-docosapentaenoic acid. Spermatocyte-enriched fractions isolated from testes of immature rats had fatty acid compositions similar to those isolated from testes of mature rats. On the other hand, spermatids isolated from immature rats had a level of docosapentaenoic acid which was intermediate between the level found in spermatocytes and that of spermatids from mature rats. Major phospholipid classes and the triacylglycerols of spermatids contained much more of the docosapentaenoic acid than the corresponding lipid types from spermatocytes. Differences in content of total phospholipids, individual classes of phospholipids and triacylglycerols among spermatocytes, spermatids and late spermatids were also observed.