A technique for the cnnultion nd perfusion of isolted rt epididyml ft pd R. J. HO nd H. C. MENG Deprtment of Physiology, Vnderbilt University School of Medicine, Nshville, Tennessee SUMMARY A method for study of the rt epididyml ft pd in vitro is described, in which perfusion vi the internl spermtic rtery is employed. The time intervl between the stoppge of the blood flow nd the estblishment of the perfusion is less thn 30 sec. Observtions of perfusion pressure, flow rte, tissue edem, nd oxygen consumption indicte tht this system cn be used to study the metbolism of dipose tissue. The sensitivity of the method with respect to free ftty cid relese stimulted by epinephrine nd ACTH is greter thn tht of incubted tissue, probbly becuse of the increse in surfce re of the ft cells exposed to the medium T H E SOLATED rt epididyml ft pd hs been used extensively for the study of lipid metbolism (1-3). n the methods employed to dte, either the whole ft pd or pieces thereof hve been incubted in vitro in suitble medi. Under these conditions, however, only the surfce of the tissue is in contct with the medium nd it would pper possible tht the metbolic function of deeper lying cells my be limited nd modified by reltively poor trnsfer of substrtes, products, nd hormones. With this in mind, we hve devised perfusion system in order to fcilitte trnsfer of substnces between medium nd cells throughout the tissue. The system hs been tested with regrd to perfusion pressure, flow rte, tissue edem, nd oxygen consumption. The relese of free ftty cids (FFA) under the influence of epinephrine nd ACTH hs been compred wi,th tht obtined by the conventionl incubtion method. We hve presented preliminry report of this work (4). A perfusion system for prmetril ft tissue hs recently been reported by Robert nd Scow (5). METHODS AND MATERALS The epididyml ft pd is usully supplied by the epididyml brnch of the internl spermtic rtery (6). 203 The internl spermtic rteries leve the ort short distnce below the left renl rtery, lthough occsionlly they brnch off from the renl rteries (Fig. 1). On the bsis of this ntomicl reltionship, it is possible to perfuse the ft pd s described below. Technique of Cnnultion Albino rts of the Sprgue-Dwley strin weighing 150-200 g were used. All nimls were fed Purin rt chow d lib. The niml ws nesthetized with Nembutl (50 mg/kg of body weight intrperitonelly), nd heprin (5 mg/kg) ws dministered intrcrdilly or intrvenously to prevent cogultion of the blood. n lter experiments heprin dministrtion ws found to be unnecessry. An incision ws mde long the line lb. After ligtion of the right spermtic rtery (ligtion in Fig. l), the left perirenl ft ws seprted nd the left kidney lifted up nd pushed to the right so s to expose the underlying ort. The ort ws clered of enveloping tissues nd ligted bove nd s close s possible to the lower lumbr rteries (ligtion 1 in Fig. 1). A loose loop ws mde round the ort just below the origin of the left renl nd upper lumbr rteries. The ort ws clmped bove the celic rtery. A smll incision ws mde, nd No. 18 or 20 needle ws inserted s cnnul nd tied (ligtion 11 in Fig. 1). At this point, the left spermtic rtery is the only remining outflow from the tied ortic segment. Thus, it is functionlly cnnultion of the left internl spermtic rtery. t is importnt to note tht occsionlly the lower lumbr rteries leve the ort t the sme level s the spermtic rtery. f this is the cse, they cn usully be tied off by plcing the ligture 1 t n oblique ngle. Perfusion ws then begun with buffered medium t pressure of bout 100 mm Hg in order to wsh the blood out of the intrvsculr comprtment. A uniform
iolumbr nt sperm.v. 8 brnch ---d---- Testiculr v.--- - - Epididyml brnch ---------- Epididymis 1 2 cm 3x FG. 1. Digrm showing the ntomicl reltionships in the cnnultion of the ort for perfusion of the rt epididyml ft pd. The order of ligtions is indicted by romn numbers. disppernce of color in the ft pd nd the testis ws tken s n indiction of stisfctory preprtion. The time required from the clmping of the ort to the beginning of perfusion ws bout 30 sec. The whole tissue mss, including testis, epididymis, left internl spermtic rtery, nd the segment of ort, ws removed from the rt by lifting up the vrious prts nd cutting the underlying connective tissue ttchments. The testiculr rtery ws then ligted between the epididymis nd testis, nd the ltter removed. n lter experiments, it ws found more desirble to plce the ligtion bove the epididymis (ligtion V in Fig. 1) nd to remove this tissue s well. The preprtion ws then plced in beker contining sline t room temperture to wsh off ny externl blood. The ft pd ws then redy for use. n some cses, the left internl spermtic rtery brnches off directly from the left renl rtery. When this ntomicl reltionship ws encountered the right ft pd ws used for perfusion. n some experiments both the rtery to nd the vein from ft pd were cnnulted. The principle for the functionl cnnultion of the internl spermtic vein ws the sme s tht of the rtery; this ws to mke the left internl spermtic vein the only inflow to the inferior ven cv which hd been cnnulted. n this cse, double ligtions were mde t the rectl level of the intestine bout /t inch from the nus nd the intestine ws severed between the ligtures. The cephlic end of the intestine ws seprted from the mesentery up to the superior mesenteric rtery in order to expose the veins in the interrenl region. The ort nd inferior ven cv were crefully seprted nd two loose loops were pplied for further cnnultions: one loop ws to secure the ortic cnnul (ligtion 111 in Fig. l), nd the other (not shown in Fig. 1) ws n oblique loop round the ven cv nd left renl vein with which the venous cnnul ws ligted. For stoppinq the venous inflow other thn vi the left internl spermtic vein, the right internl spermtic vein wzs ligted with its ccompnying rtery (ligtion ) nd the ven cv ws tied with the ort (ligtion 11) s shown in Fig. 1. The left renl vein ws lso ligted close to the kidney, nd the brnch of the left internl spermtic vein ws lso tied. After clmping the ven cv t the right renl level, n incision ws mde below it. Heprinized polyethylene tubing ws inserted until the tip reched the junction of the left renl vein nd the ven cv; the loop ws then secured. After both vein nd rtery hd been cnnulted, the ligtion V ws mde. The oxygen consumption of the ft pd ws determined by the rteriovenous difference in the oxygen tension from doubly cnnlted preprtion. The oxygen tension ws mesured by the method of Sproule et l. (7). Perfusion of the Epididyml Ft Pd The wshed ft pd ws then trnsferred to the perfusion pprtus. Two vrints were used. The first ws employed when recircultion of smll volume of medium through the tissue ws desired. n this system, the cnnul ws connected to n pprtus modified from tht described by Morgn et l. (8) for perfusion of the rt hert. The medium ws pumped through the ortic cnnul into the ft pd, which rested on sintered glss filter disk in the thermosttic tissue chmber. Medium left the tissue through the cut brnches of the internl spermtic vein nd returned to the tissue chmber (Fig. 2). The perfuste then pssed through the filter nd ws recirculted. The flow rte ws estimted by counting the drops flling into the bubble trp (Fig. 2). The second system ws used for perfusion without recircultion, the so-clled open system. The medium ws contined in thermosttic reservoir consisting of wter-jcketed condenser of bout 60 ml cpcity. t ws equilibrted with 95% 0 ~ 5 % C02 by gentle continuous bubbling. The outflowing medium pssed through the peristltic pump, the bubble trp, nd the ft pd locted in the tissue chmber s shown in Fig. 2 nd ws collected in 15-ml grduted conicl centrifuge tubes. The flow rte ws estimted by counting either 204 JOURNAL OF LPD RESEARCH VOLUME 5, 1964
the drops flling from the top of the bubble trp or those from the tissue chmber into the collecting tube. n both types of perfusion system the flow rte ws mintined t bout 0.5 ml/min by djusting the pressure developed by the peristltic pump. The first 5 ml of perfuste contining red blood cells were discrded nd the perfuste collected therefter ws cler. A three-wy stopcock ws inserted between the bubble trp nd the tissue chmber just bove the cnnul. This provided convenient route for the ddition of hormones in the recircultion experiments, nd for chnging from one type of medium to nother in the open system. The perfusion medium ws Krebs-Ringer phosphte or bicrbonte buffer contining 5% bovine serum lbumin s FFA cceptor (9). Determintions of FFA nd Tissue Protein ivitrogen Free ftty cid content of the medium ws determined by the method of Dole (lo), modified to use Nile Blue A s n indictor in the titrtion mixture. Tissue FFA ws determined by the sme procedure except tht the tissue ws homogenized in the extrction mixture. A segment of the tissue, weighing bout 100 mg, ws homogenized in 2 ml of sline nd centrifuged. The underlying cler lyer ws used for the soluble protein nitrogen determintion ccording to Lowry (1 1). Experiments with il onperfused Ft Pds Whole ft pds or segments of them were quickly weighed on torsion blnce nd then incubted in medium contining epinephrine in Dubnoff metbolic shker t bout 100 cycle/min. The incubtion time, volume, nd other conditions re indicted under individul experiments. The FFA content in medium nd tissue ws determined fter incubtion ccording to methods described bove. Mterils Adrenlin chloride, 1 mg/ml in physiologicl sline, ws supplied by Prke, Dvis nd Co., Detroit, Mich. Lyophilized ACTH, lot No. 116283, ws supplied by Wilson Lbortories, Chicgo, ll.; 1 mg is equivlent to 100 units. Crystllized bovine serum lbumin (lbumin bovine crystlline 2X) ws obtined from Nutritionl Biochemicls Corp., Clevelnd, Ohio. n some experiments, bovine serum 1 bumin ws extrcted ccording to the method of Goodmn (12) with slight modifiction ; this preprtion hs been designted s FFA-free serum lbumin. RESULTS n hydrodynmic system, the flow rte depends on the perfusion pressure, the tissue resistnce, nd the viscosity of the circulting medium. f the flow rte nd the viscosity of the medium re constnt, the pressure chnge is proportionl to the tissue resistnce. Perfusion of the rt epididyml ft pd ws crried out on the bsis of this reltionship. At the beginning of the preliminry wshing by perfusion, the pressure ws bout 100 mm Hg. During the first 5 min the pressure declined slowly nd becme reltively stedy t between 25 nd 75 mm Hg (vrying from one pd to nother). The pressure in 36 experiments ws mintined t n verge of 52 *0.13 mm Hg during 60 min perfusion period. The flow rte during perfusion ws kept constnt t bout 0.4-0.5 ml/min. Fig. 3 represents the verge of two experiments illustrting the time course of perfusion pressure nd flow rte. Stedy sttes in perfusion pressure nd flow rte were lso mintined during the 50 min experimentl period when epinephrine (0.03 pg/ml) ws employed. Oxygen Consumption The oxygen tension of the medium ws determined before nd fter perfusion. Tht of the rteril smple tken from the medium reservoir during perfusion ws Wter jocket- -.. ~~ Bubble trp - - - - Peristltic PUPD - 3 wy stopcock - 95% 0, - 5 70 GO2 Tissue chmber----- Wter jcket--- --- - Segment of Aort -nternl Sperm. -Supporting disc FG. 2. Digrmmtic representtion of the pprtus for perfusion of the ft epididyml ft pd. The digrm shows the recircultion of medium during perfusion. A smll mount of medium usully collects bove the filter disk nd the gs inlet dips into this fluid; the continuous bubbling fcilittes gs exchnge. When n open circuit (non-recircultion) perfusion is crried out the filter disk is replced by corsely perforted teflon plte. Ho AND MENG Cnnultion nd Perfusion of Rt Epididyml Ft Pd 205
bout 505 mm Hg throughout the experiment. The venous smple ws obtined directly from the venous cnnul nd collected into gs-seled syringe. t verged 213 mm Hg t the bsl stte, with rnge from 180 to 262 mm Hg. n the presence of epinephrine (0.01 pg/ml) plus glucose (100 mg/100 ml) the oxygen tension decresed to n verge of 100 mm Hg; the lowest finding ws 85 mm Hg. The men tissue oxygen consumption ws 386 pl/g per hr under the bsl stte conditions, nd 561 pl/g/ per hr when epinephrine nd glucose were dded (Tble 1). Weight Gin of Epididyml Ft Pd During Perfusion n the erly recircultion experiments, pressure of 100 mm Hg or higher ws mintined. Following prolonged perfusion t this pressure gross edem ws usully observed. The edem ws less pronounced in ft pds perfused t low pressure (40-50 mm Hg). Tble 2 shows the verge weight gin of perfused ft pds with pressure mintined t bout 50 mm Hg nd flow rte t 0.4-0.5 ml/min. Protein Nitrogen Content of the Perfused Ft Pds The protein nitrogen content of the epididyml ft pds vried with the nutritionl stte of the niml nd the mount of the plsm protein nitrogen trpped in the tissue. Tble 3 shows the protein nitrogen contents of epididyml ft pds from fed nd fsted rts both CT 3 2 80 before nd fter wshing by perfusion with bicrbonte buffer. Comprison of FFA Relese from the Perfused nd ncubted Ft Pds Figure 4 shows tht the perfused ft pds were more sensitive thn the incubted tissues in respect to FFA relese. The verge net production of FFA from the perfused ft pds ws 2.5 times s much nd the FFA relese ws 11 times s much s tht of the incubted tissues. The verge tissue:medium FFA rtio of the incubted ft pds ws 9, while tht of the perfused ft pds ws bout 1.5. Efed of Prior njection of Heprin on the FFA Relese nduced by Epinephrine Although prior dministrtion of heprin ws not necessry, it did fcilitte considerbly the wshing by perfusion of the ft pd. Since heprin is known to induce the relese of the clering fctor lipse, the r 6.03t: - 0 E 0 O 20 30 40 50 TME (min 1 FG. 3. The time course of perfusion pressure nd flow rte during 50 minute perfusion period. Ft pds from fed rts were perfused with Krebs-Ringer bicrbonte buffer ph 7.4 contining 5 yo FFA-free bovine serum lbumin. Perfusion ws crried out without recircultion for 10 min, fter which medium contining epinephrine (0.03 pg/ml) ws used. The results re verges of two representtive experiments. 0-0 bsl medium without epinephrine; 0-4 bsl medium with the ddition of epinephrine (0.03 Mg/ml). 206 JOURNAL OF LPD RESEARCH VOLUME 5, 1964 FG. 4. Comprison of the increses in the FFA content of tissue nd medium of the perfused nd incubted epididyml ft pds of fed rts. The perfusion or incubtion period ws 30 min. A Krebs-Ringer phosphte buffer ph 7.4, contining 5pg of epinephrine per ml nd 5 % bovine serum lbumin ws used s medium for both incubtion nd perfusion experiments. The recircultion perfusion system ws used. The ft pd of incubtion ws dissected, weighed nd incubted in n Erylenmeyer flsk contining 3.0 ml of buffer. fs4 incubtion; perfusion. Figures in prentheses represent the number of ft pds. The verge FFA content of the ft pds from 23 fed rts before incubtion ws 0.44 f 0.004 pmole/mg of N. FFA content nd tissue nitrogen befpre incubtion were determined on the other ft pd of the sme rt nd the ltter vlue ws used to express the units of FFA in the tissue nd medium. n these units verge totl FFA produced by the incubted nd by the perfused ft pds ws 3.91 nd 10.47 pmole/mg of N reprectively. The totl FFA produced thus clculted is ctully the net FFA produced under the described experimentl conditions since the quntities of FFA relesed by lipolysis nd utilized for reesterifiction during incubtion or perfusion were not known.
~ ~ ~~~ ~ ~ TABLE 1 OXYGEN CONSUMPTON OF THE SOLATED PERFUSED EPDDYMAL FAT PADS* ~~ ~ AOz t Bsl Medium Perfusion Epinephrine Plus Glucose Perfusion Tissue Before After Flow 025 Flow 01 Wt Perfusion Perfusion VO1$ A02 - VOt Rte Consumption VOz A02 - V02 Rte Consumption ms mm Hg mm Hg mm Hg mm Hg ml p/fid/hr pl/g/hr mm Hg mm Hg ml p/fid/hr pllglhr 660 504 501 180 325 0.4 251 379 85 420 04 325 490 626 506 184 321 0.4 248 394 108 397 0 5 384 610 700 206 299 0.5 288 412 100 405 0.5 391 559 650 262 243 0.5 234 358 108 397 0.5 383 585 * Epididyml ft pds from fed rts were doubly cnnulted nd perfused with Krebs-Ringer bicrbonte buffer, ph 7.4, contining 5% FFA-free bovine Berum lbumin first without nd then with epinephrine (0.03 pg/ml) nd glucose (100 mg/loo ml) for 15 min ech. The rteril perfuste ws tken from the medium reservoir nd the venous perfuste ws collected in gs-seled syringe for pot ssy. t Oxygen tension of perfuste from the reservoir. 3 Oxygen tension of venous perfuste. 5 The tissue oxygen consumption ws clculted by multiplying the A - V oxygen difference( bsed on the oxygen solubility in wter) by the flow rte nd dividing by tissue weight. Mens f SE: 386 f 12 pg/g/hr (bsl), 561 f 26 pg/g/hr (epinephrine). TABLE 2 WEGHT GAN OF EPDDYMAL FAT PADS DURNG PERFUSON No. of Perfusion Observed Averge Expts. Time Weight Gin Wter Content min % % 4 0 0 16.3 f 3.71 6 30 15.9 f 6.6 27.7$ 6 45 16.3 f 4.0 27.8t 6 60 18.0 f 3.0 28.83 * Ft pds from fedrts were perfused with Krebs-Ringer bicrbonte buffer, ph 7.4, contining 5% FFA-free bovine serum lbumin. Wet weight of ech ft pd ws obtined by weighing in torsion blnce before nd fter perfusion. The ssumption ws mde tht the weight of cnnul nd the segment of ort were constnt throughout the perfusion period. $The verge wter content of ft pds before perfusion ws estimted by difference between the weights of wet nd dry hue. $Wter content (%) of the ft pd fter perfusion ws clculted ccording to the following formul : Gin in wet wt during perfusion (mg) + Wter content before perfusion (mg) Wet wt fter perfusion (mg) x 100 possibility of n effect of heprin on the epinephrineinduced FFA relese ws tested. Results re shown in Tble 4. t cn be seen tht no significnt difference in FFA relese between the two groups ws observed. Tim Course of FFA Relese nduced by Ep nephrine nd ACTH The time courses of FFA relese induced by continuous perfusion with ephephrine nd ACTH re shown in Figs. 5A nd 5B respectively. Epinephrine (0.03 pg/ml) nd ACTH (100 pu/ml) both stimulted the FFA relese from ft pds. The Ability of the PMfwed Ft Pd to h pond to Repefed Stimultion n this study, two equl stimultions of short durtion were pplied; the second stimultion ws given fter the tissue hd recovered from its reponse to the first. The * Ho AND Mmo TABLE 3 SOLUBLE PROTEN NTROGEN CONTENT PERFUSED AND THE NONPERFUSED FAT PAD OF THE No. of Before After Expts. Condition Perfusion Perfusion Decrese mg N/g mg N/g % 5 Fed 2.68 f O.Ob 1.65 4z 0.04 39 10 F.isted 36 hr 3.25 f 0.40 2.24 f 0.70 31 Ft pds were perfused with Krebs-Ringer bicrbonte buffer, ph 7.4 (no lbumin) for 10-20 min until the perfuste ws cler nd free of blood cells. TABLE 4 EFFECT OF PROR NJECTON OF HEPARN FFA RELEASE NDUCED BY EPNEPHRNE ON THE No. of Pretretment Time of Perfusion Expts. with Heprin with Epinephrine FFA Relese min Wolclmg of N 13 + 30 4.89 f 0.27* 5-30 4.25 f 0.84 Ft pds of fed rts were used in ll experiments; epinephrine concentrtion ws 0.03 fig/ml. Heprin (5 mg/kg) ws injected intrcrdilly or intrvenously before. surgicl procedures. Ft pds were perfused with Krebs-Ringer bicrbonte buffer, ph 7.4, contining 5% FFA-free bovine serum lbumin in nonrecircultion system. *Men f SE. results of double stimultion by epinephrine re shown in Fig. 6. The mounts of FFA relesed following both stimultions were identicl. DSCUSSON n studies in which orgns re perfused the oxygen supply, perfusion pressure, flow rte, nd tissue edem should lwys be considered. The oxygen supply of the perfused ft pd in the present work seems dequte since the oxygen tension of the rteril perfuste in the medium reservoir ws mintined t high nd constnt level throughout the experimentl period. Furthermore, lthough the oxygen tension of the perfuste collected Cnnullion nd Perjuwn of Rt Epididyml Ft Pd 207
fter perfusion of the tissue ws 55% lower thn tht of the rteril perfuste, it ws higher thn tht of the venous or even rteril blood of norml niml (13); the results suggest tht there is n dequte oxygen supply for tissue consumption. This is true even in the presence in the perfusion medium of epinephrine, which incresed the oxygen consumption by 46% bove the bsl stte. The oxygen consumption of the perfused ft pds determined in the present study ws higher thn tht reported by Hgen et l. using incubtion techniques (1 4). The fll in pressure during the preliminry wshing by perfusion might hve been due to the chnge of viscosity from blood to the buffer medium. Differences in tissue resistnce might hve cused the vrition of pressure between individul ft pds during perfusion. How- 0.3r 0.oL - %j 0.3- W Z -1 : E 0.2-0.1 A 6 --- / Bsl Medium? Hormone Time (min 1 FG. 5. The time course of FFA relese from ft pds induced by epinephrine (A) nd by ACTH (B). Ft pds were perfusionwshed with Krebs-Ringer bicrbonte buffer ph 7.4 contining 5% FFA-free bovine serum lbumin until the venous perfuste ws free of red blood cells. Smples of perfuste were collected t 5- to O-min intervls. After collecting the control smples the medium contining epinephrine (0.03 fig) or ACTH (100 pu/ml) ws introdced. The control groups were perfused with the sme bsl medium without the hormone throughout the experiment. Fed rts were used for investigting epinephrine effects (A) nd fsted (36-48 hr) rts for exmining the effect of ACTH (B). O-----O FFA relese induced by epinephrine (A) or by ACTH (B)..-----O FFA relese of the control group. 208 JOURNAL OF LPD RESEARCH VOLUME 5, 1964 1.- c 0-41 W cn W Slimulotion Epinephrine Stimultion TME (MN FG. 6. The response of the perfused ft pd to repeted stimultion by epinephrine. Ft pds from fed rt were first perfused with the bsl medium, Krebs-Ringer bicrbonte buffer contining 5% FFA-free bovine serum lbumin, ph 7.4, for 6 min to collect 3 smples t 2-min intervls. After this the perfusion ws crried out with medium contining epinephrine (0.1 pg/ml) for 2 min by switching the 3 wy stopcock loctedibetween the tissue chmber nd the bubble trp; t the end of the 2 min, epinephrine-free bsl medium ws gin used. Smples of perfuste were collected t 2-min intervls. The second stimultion ws given fter the FFA level hd fllen to the initil level. Nonrecircultion perfusion ws used. Verticl lines represent the stndrd errors. Ech point represents the verge of 4 pds. ever, in the stedy stte, even when the blood ws completely replced by the perfusion medium, occsionl minor djustments of the pump were necessry in order to mintin the flow rte constnt t bout 0.5 ml/min. The reson for using 0.5 ml/min rther thn higher rte ws tht less pressure ws required to mintin this rte nd, therefore, less edem of the tissue developed. The presence of some degree of edem did not pprently interfere with the FFA relese from the ft pd. Furthermore, the wter content remined firly constnt fter the initil rise. This perfusion flow rte of the reltively nonviscous medium is bout 5 times higher thn tht of the blood estimted in situ. The decrese in tissue protein nitrogen fter wshing by perfusion is believed to be due to the loss of plsm protein from the vsculr comprtment of the tissue, nd indictes the importnce of this preliminry step in the study of FFA mobiliztion, since plsm FFA is known to be bound to serum lbumin. The increse in sensitivity in FFA relese from the perfused ft pd is probbly due to the fct tht the medium ws supplied by physiologicl route which increses the functionl surfce between the medium nd ft cells. According to Winegrd every ft cell of the epididyml ft pd is surrounded by cpillries nd every cpillry is surrounded by ft cells (15). The 1 R. J. Ho nd 1. C. Meng, unpublished dt.
volume of the vsculr comprtment of dipose tissue is comprble with tht of muscle (16). Thus, the surfce re of ft cells exposed to the perfuste is much lrger thn tht exposed to the incubtion medium. Furthermore, the mixing nd the entering into cells of the substrtes nd hormones in the medium nd the removing of the products from cells re probbly fcilitted by the perfusion process. t is evident tht the isolted perfused dipose tissue remins in functionl stte nd responds continuously to the stimultion of epinephrine ; it lso responds eqully to the repeted stimultion. n fct, perfused ft pds hve been mintined in good functionl condition for s long s 100 min.1 Prior dministrtion of heprin s n nticogulnt to rts seems to hve no effect on the FFA mobiliztion induced by epinephrine. n fct, even the ddition of heprin to the perfusion medium did not modify the FFA relese induced by epinephrine from the epididyml ft pd.* t is concluded tht the methods of cnnultion nd perfusion of rt epididyml ft pd meet the bsic requirements used s criteri in perfusion systems. Furthermore, this technique would be useful in mny spects in which the current incubtion method is not stisfctory. The uthors wish to express their pprecition to Dr. Chrles R. Prk for his invluble suggestions nd continued interest throughout this work. The ssistnce of Dr. Howrd E. Morgn in the development of the perfusion pprtus nd of Dr. Lloyd H. Rmsey for the mesurements of oxygen tension re lso pprecited. * R. J. Ho, S. J. Ho, nd H. C. Meng, unpublished dt. This investigtion ws supported by PHS Reserch Grnt HE-04372-03 from the Ntionl nstitutes of Helth, U. S. Public Helth Service ; the Americn Hert Assocition ; nd the Ntionl Science Foundtion (G-23770). A description of this work ws submitted (by R. J. H.) to the Fculty of the Grdute School of Vnderbilt University in prtil fulfillment of the requirements for the degree of Doctor of Philosophy. Mnusrript received August 5, 7963; ccepted November 4, 7963. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. REFERENCE s Vughn, M. J. Lipid Res. 2: 293, 1961. Jenrenud, B. Metb., Clin. Exp. 10: 535, 1961. Wertheimer, E., nd E. Shfrir. Recent Progr. in Hormone Res., 16: 467, 1960. Ho, R. J., nd H. C. Meng. Federtion Proc. 21: 292, 1962. Robert, A., nd R. 0. Scow. Am. J, Physiol. 205: 405, 1963. Greene, E. The Antomy of the Rt. Hfner Publ. Co., New York, 1955, p. 199. Sproule, B. J., W. F. Miller,. E. Cushing, nd C. B. Chpmn. J. Appl. Physiol., 11: 365, 1957. Morgn, H. E., M. J. Henderson, D. M. Regen, nd C. R. Prk. J. Biol. Chem. 236: 253, 1961. Cohen, P. P. n Mnometric Tcchniques, edited by W. W. Umbreit, R. H. Burris, nd J. F. Stuffer. Burgess Publ. Co., Minnepolis, 1957, p. 149. Dole, V. P. J. Clin. nvest. 35: 150, 1956. Lowry, 0. H., S. J. Rosebyrough, A. L. Frr, nd R. J. Rndll. J. Biol.Chem. 193: 265, 1951. Goodmn, Dew. S. Science 125: 1296,1957. Crbon, L. D. n Medicl Physiology nd Biophysics, edited by T. C. Ruch nd J. F. Fulton. W. B. Sunders Co., Phildelphi nd London, 1960, p. 793. Hgen, J. M., E. G. Bll, nd 0. Copper. J. Biol. Chem. 234: 781, 1959. Winegrd, A.. n Adipose Tissue s n Orgn, edited bv L. W. Kinsell. Chrles CThoms, Springfield, ll., 1962, p. 61. 16. Husberger, F. X., nd M. M. Widelitz. Am. J. Physiol., 204: 649, 1963. Ho AND MENG Cnnultion nd Perfusion of Rt Epididyml Ft Pd 209