N G,N G -Dimethyl-l-arginine,a dominant precursor of endogenous dimethylamine in rats

Summary The metabolic significance ofN ^G ,N ^G -dimethyl-l-arginine (DMA) as a precursor of endogenous dimethylamine (DMN) in rats was examined in connection with the wide distribution and active operation of dimethylargininase (EC3.5.3.18) in rat tissues (Kimoto et al., 1993). When [methyl-¹⁴C]DMA was administered intraperitoneally to rats, the radioactive DMN was detected in various tissues as a major radioactive metabolite one hour after injection, and about 65% of the radioactivity administered was recovered in the first 12-h urine as DMN. In the case of the [¹⁴C] DMN-injected rats, almost all the radioactivity was excreted in the 12-h urine as DMN, except for a negligible amount of radioactivity found in urea. The time-dependent decrease in the specific radioactivity of DMA and DMN in urine showed that dimethylargininase was significantly involved in thein vivo formation of DMN by the hydrolytic cleavage of DMA released from methylated proteins and that DMA is a dominant precursor of endogenous DMN in rats.     

Metabolism of 14C-arachidonate in rat isolated lung

Metabolism of ¹⁴C-arachidonate was investigated in rat isolated lungs perfused via the pulmonary circulation with Krebs solution. Only 10% of the radioactivity derived from an infusion of ¹⁴C-arachidonate through the pulmonary circulation of rat isolated lungs appeared in the effluent by 10 minutes. At 10 min, the major component of effluent radioactivity and 20–40% of that retained in lung was unchanged arachidonate. Between 10 and 20 min of perfusion, a further small amount of radioactivity was lost in lung effluent and at 20 min the retained radioactivity showed a decrease in the proportion present as free arachidonate. Between 20 and 60 min, there was no further loss of radioactivity in effluent and no further change in the distribution in lung. Addition of albumin to the Krebs solution perfusate during the infusion of ¹⁴C-arachidonate increased effluent radioactivity to 80%, but albumin added after 10 min only caused the efflux of a small amount of radioactivity (10%). Treatment of labelled lung at 20 min with the calcium ionophore A23187 released biologically active metabolites of arachidonate but very little radioactivity. Metabolism of arachidonate, either during the infusion or after retention in lung, in rat lung was closer to that in human lung than to that in guinea-pig lung.     

The distribution of 14C-imipramine in mice bearing Lewis lung carcinoma

The distribution of ¹⁴C-imipramine (10 mg/kg ip) and several of its metabolites in tumor, lung, liver, and kidney was investigated in male BDF₁ mice bearing Lewis lung carcinoma. In contrast to other tissues, the tumor exhibited a pronounced absorption phase of ¹⁴C-imipramine; peak concentrations were reached approximately 2 hours after administration. The lung accumulated more imipramine than other tissues at early time points; however, by 12 hours the lung had the lowest tissue/plasma ratio of ¹⁴C-imipramine-derived radioactivity of the tissues studied. In both lung and tumor, the metabolic profile of imipramine was similar, with unchanged imipramine predominating; 2-hydroxyimipramine was the principle metabolite in liver. The presence of Lewis lung tumor had minimal effects on the distribution and metabolism of imipramine.     

Metabolism of C-arachidonate in rat isolated lung

Metabolism of ¹⁴C-arachidonate was investigated in rat isolated lungs perfused via the pulmonary circulation with Krebs solution. Only 10% of the radioactivity derived from an infusion of ¹⁴C-arachidonate through the pulmonary circulation of rat isolated lungs appeared in the effluent by 10 minutes. At 10 min, the major component of effluent radioactivity and 20–40% of that retained in lung was unchanged arachidonate. Between 10 and 20 min of perfusion, a further small amount of radioactivity was lost in lung effluent and at 20 min the retained radioactivity showed a decrease in the proportion present as free arachidonate. Between 20 and 60 min, there was no further loss of radioactivity in effluent and no further change in the distribution in lung. Addition of albumin to the Krebs solution perfusate during the infusion of ¹⁴C-arachidonate increased effluent radioactivity to 80%, but albumin added after 10 min only caused the efflux of a small amount of radioactivity (10%). Treatment of labelled lung at 20 min with the calcium ionophore A23187 released biologically active metabolites of arachidonate but very little radioactivity. Metabolism of arachidonate, either during the infusion or after retention in lung, in rat lung was closer to that in human lung than to that in guinea-pig lung.     

Concentration of 3-Methylindole (3MI) and distribution of radioactivity from 14C3MI in goat tissues associated with acute pulmonary edema

3-Methylindole (3MI) can cause acute pulmonary edema in goats. Because of known lipophilic properties and direct effects on biological membranes, the concentration of 3MI and distribution of radioactivity from ¹⁴C3MI in tissues was investigated during development of 3MI-induced APE. Goats were given a 2 hr jugular infusion of 3MI containing ¹⁴C3MI using propylene glycol as the vehicle. Groups of 3 goats were killed at 0, .5, 2 and 4 hr and 2 goats were killed at 8 and 24 hr. Plasma, lung, liver, kidney and other selected tissues were collected. 3MI was rapidly cleared from blood plasma and tissues after infusion, and 81% of the radioactivity was excreted in the urine by 24 hr. Maximum concentrations of unmetabolized 3MI in the tissues ranged from 2.6 to 15 μg 3MI/g, including 7.5 μg 3MI/g in the lung. The ratios of equivalent radioactivity to unmetabolized 3MI indicated rapid metabolism and the presence of metabolites in all tissues studied. The lung contained the highest proportion of metabolites with ratios of radioactivity to unmetabolized 3MI of about 50, 10, 250, 150 and 80 at 0.5, 2, 4, 8 and 24 hr. The data demonstrate that 3MI does not selectively concentrate in the lung and that the concentrations are lower than those usually associated with direct membrane damage. They also indicate that 3MI is rapidly metabolized and that metabolites are present in tissues, especially the lung. These results suggest that direct effects of 3MI on biological membranes are not primarily responsible for lung injury in goats.     

Suppression of liver uptake of orally fed liposomes by injection (I.P.) of dextran sulfate 500

¹²⁵Iodine labelled human immunoglobulin-G encapsulated liposomes were administered orally to rats. Distribution of radioactivity was checked in various tissues and in portal blood. The effect of dextran sulfate (DS 500,000 m. wt., liver blockade agent) injection (i.p.) on the liver uptake of liposomes and on the amount of liposomes appearing in the portal blood from the gastrointestinal tract have been studied. An increased amount of radioactivity was observed in the portal blood and the amount of radioactivity in the liver decreased appreciably after injection of dextran sulfate. In both the cases the action of dextran sulfate started 2 hours after injection and reached maximum at 12 hour, falling slightly at 24 hour.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.     

Distribution of i.v. administered epidermal growth factor in the rat

The distribution of i.v. injected ¹²⁵I-labeled epidermal growth factor (EGF) was examined in the rat. The uptake of radioactivity was examined for the following tissues: liver, kidney, skin, stomach, small intestine, colon, brain, submandibular gland, lung, spleen, and testis. ¹²⁵I-EGF was cleared from the circulation within minutes. At 2.5 min after the injection only 7% of the label was left in the blood. Most of the label was found in the liver (52%), the kidneys (14%), the small intestine (11%) and the skin (7%). The other organs examined contained 1% or less of the radioactivity. The uptake of ¹²⁵I-EGF per g tissue was markedly higher for the liver and kidneys than for the rest of the organs. By autoradiography ¹²⁵I-EGF was found in the peripheral parts of the classical liver lobule, in the proximal tubules of the kidneys, in the surface epithelium of the stomach, and in the surface epithelium of the villi in the small intestine. In conclusion the present study showed that small doses of homologous EGF was cleared from the circulation of rats within minutes, mainly by the liver, the kidneys, and the small intestine.     

Studies on distribution and metabolism of valproate in rat brain,liver, and kidney

Time-course studies on the distribution and metabolism of valproate (VPA) in rat brain, liver, and kidney, after intraperitoneal injection of a mixture of [¹⁴C]VPA and [³H]VPA, showed that: (1) maximal amount of radioactivity in the various tissues was observed after 30 min from the time the drug was administered; (2) at 30 min the distribution of labeled VPA in brain, liver, and kidney was 17%, 64%, and 19% of the total radioactivity, respectively; (3) at 24 hr more than 97% of the total radioactivity was lost from the tissues and the¹⁴C/³H ratios increased significantly with time. Studies on the regional distribution of the drug showed that it is relatively homogeneously distributed. Studies on the subcellular distribution of the drug showed that it is associated mostly with the soluble and mitochondrial fractions, with little radioactivity in the myelin and synaptosomal fractions. Radiochromatography of VPA metabolites in perchloric acid extracts from brain, liver, and kidney revealed the presence of four metabolites. VPA was not incorporated into phospholipids of the neuronal membranes. Furthermore, it had no significant effects on Mg²⁺-ATPase and (Na⁺+K⁺)-ATPase in synaptosomes and microsomes obtained either from control or from rats injected with VPA. It was concluded that this antiepileptic drug does not appear to act through its incorporation into neuronal membrane or through its action on the Na⁺ pump.Contribution No. 0601 from the Department of Cell and Molecular Biology, the Medical College of Georgia, Augusta, Georgia 30912.     

In vivo assessment of 6-deoxy-6-[18F]fluoro-d-galactose as a PET tracer for studying galactose metabolism

The potential of 6-deoxy-6-[¹⁸F]fluoro-d-galactose (6-[¹⁸F]FdGal) as an in vivo tracer for studying galactose metabolism in tumors and liver was investigated. High uptake and rapid clearance of the radioactivity were observed in many organs of mice after i.v. injection of the tracer. d-Galactose loading did not affect liver uptake. Three experimental tumors showed a slightly higher uptake than other tissues, and rat brain tumor was clearly visualized by autoradiography. However, the radioactivity in tumors decreased rapidly. In the liver, a significant amount of the tracer was found in a galactonate form, while this oxidation was a minor metabolic pathway in the tumors. In both tumor and liver tissues, small amounts of the tracer were incorporated into macromolecular glycoconjugate via phosphate and uridylate forms as intermediate precursors. These results indicate that 6-[¹⁸F]FdGal is not suitable for studying galactose metabolism in vivo because of the low affinity of the tracer for the metabolism.     

Dopamine β-hydroxylase: Determination of half-life and appearance in lymph fluid after IV injection of 125I-DBH into rats

A 4 day half-life of dopamine beta-hydroxylase (DBH) was determined for rats injected IV with ¹²⁵I-rat DBH from the slow exponential component of radioactivity appearing in plasma, urine, feces and combined urine and feces. Half-life estimates for ¹²⁵I-rat DBH injected IV into WKY and SHR animals did not differ from Sprague Dawley (Zivic Miller) rats. Radioactivity declined in parallel in plasma, urine and feces following IV ¹²⁵I-rat DBH administration and each radioactivity falloff curve could be resolved into two components. The slow phase of the decline of radioactivity excreted into urine and feces from which DBH half-life was calculated occurred between 5 and 25 days after ¹²⁵I-rat DBH injection. The early fast phase which is associated with distribution of the exogenous protein in body fluids and tissues continued for approximately the first 140 hr after DBH injection. The distribution characteristics of IV administered active bovine DBH and ¹²⁵I-rat DBH into the lymphatic system were examined. After active bovine DBH or ¹²⁵I-rat DBH was injected IV into rats, active DBH or radioactivity, respectively, appeared in lymph fluid (thoracic duct) within 20 min; reached peak concentrations within 90 min, and thereafter, declined in parallel with the plasma concentration. The concentration of radioactivity in plasma and lymph fluid were found to be unequal at 9 hr but were equivalent 68–75 hrs after IV injection of ¹²⁵I-rat DBH. Based on the amount of active DBH or radioactivity which accumulates in lymph fluid it is clear that'a substantial amount (> 50%) of the DBH in blood circulates through the lymphatic channels. Analysis of parallel experiments with labelled serum albumin indicate that use of these methods to study plasma proteins do provide sensitive measures of biological half-life and lymphatic distribution characteristics. Specifically for DBH, the results of our study suggest that DBH normally circulates in plasma and lymph fluid with a biological half-life of 4 days.     

Evidence for binding of metabolically activated 1,2-dibromoethane to chromatin of forestomach and liver

The covalent binding of metabolically activated 1,2-dibromoethane (DBE), a potent carcinogen, to chromatin constituents of forestomach and liver was examined $\text{in vitro}$. Chromatin was prepared from forestomach and liver of B6C3F1 mice and characterized. In order to activate DBE, microsomes and cytosol were isolated from mouse forestomach and liver and incubated with [¹⁴C]-DBE in the presence of a NADPH regenerating system. Results demonstrate that DBE bound covalently to the same extent to protein of microsomes and chromatin isolated from forestomach and liver. On the contrary, DBE bound significantly more to chromatin DNA of forestomach or liver than it did to salmon sperm DNA. It appears from these results that the metabolically activated DBE is more reactive to homologous DNA than exogenous DNA. Fractionation of DBE-bound chromatin protein into histone and nonhistone proteins resulted in higher binding of DBE to non-histone than to histone proteins isolated from forestomach and liver.     

Differences between pancreatropic nitrosamine carcinogens and N-nitrosodimethylamine in methylating DNA in various tissues of hamsters and rats

N-Nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl)(2-oxypropyl)amine (HPOP) induce pancreatic tumors in the Syrian hamster. BOP and HPOP target the kidneys, esophagus and upper respiratory system in rats, but the pancreas of this species is resistant to the above carcinogens. On the other hand, N-nitrosodimethylamine (DMN) induces hepatic and kidney tumors in the rat, and tumors of the liver and upper respiratory system in the hamster, but it is not known to affect the pancreas of either species. At equimolar doses, ratios of DMN versus BOP or HPOP mediated methylation in hamster liver DNA are 1.6 and 8.1, respectively. Respective ratios in the rat liver are 1.1 and 6.5. However, in both species equitoxic doses of BOP, HPOP and DMN induce similar levels of N7-methylguanine (N7-MeG) in hepatic DNA. At such doses methylation of kidney DNA is 24 and 14 times more extensive in BOP and HPOP than in DMN-treated hamsters. Similarly, ratios of N7-MeG in the pancreas of BOP and HPOP vs. DMN-treated hamsters are 10 and 5, respectively, while in the lung this ratio is 2.2 for both carcinogens. Levels of O6-methylguanine (O6-MeG) in the DNA of extrahepatic tissues are substantially greater in hamsters treated with BOP or HPOP than in those treated with an equitoxic dose of DMN. In rats, equitoxic doses of BOP and DMN induce similar levels of N7-MeG and O6-MeG in hepatic, kidney and lung DNA. However, levels of these adducts in pancreatic DNA are 2 times greater following BOP than DMN administration. Ratios of N7-MeG in pancreas, lung and kidney in HPOP vs. DMN-treated rats are 2.1, 2.7 and 2.1, respectively. Repair of O6-MeG is more effective in rat than in hamster liver, however in other tissues this is not always the case. Levels of O6-MeG in the pancreas of rats are reduced to half of their initial value between 40 and 50 h following the administration of 10, 50 or 20 mg/kg DMN, HPOP or BOP, respectively. However, half-lives for the repair of O6-MeG in hamster pancreas are 28, 62 and greater than 120 h at the respective doses of the above carcinogens. Since the above doses of DMN, HPOP and BOP induce 7, 19 and 41 nmol O6-MeG/mmol of guanine respectively in the hamster pancreas, it is suggested that the rate of repair could be a function of the initial concentration of this adduct. Differences between DMN and BOP or HPOP in methylating pancreatic DNA are sufficient to distinguish the latter two nitrosamines as pancreatic carcinogens for the hamster.     

Axonal transport in nigro-neostriatal neurons during morphine tolerance development and abstinence in rats.

Axonal transport of [³H]protein in the nigro-neostriatal pathway in rats was examined during acute and chronic morphine administration and during morphine abstinence. Two days after a microinjection of [³H]lysine into the left substantia nigra zona compacta, more than 95% of the radioactivity present in the rat forebrain was protein-bound. Examination of frozen frontal brain sections revealed that 80–90% of the labelled protein of the injected side was located in brain areas traversed by the nigro-neostriatal pathway. As a positive control, intranigrally administered colchicine reduced the amount of [³H]protein transported after 5 days to the nucleus caudatus-putamen (neostriatum) to approx 18-26% of control. In animals rendered morphine-dependent by subcutaneous implantation of tablets containing 75 mg of morphine base, 27–86% more radioactivity accumulated in the neostriatum at 3, 4 and 5 days after [³H]lysine injection. In contrast, 23–48% less radioactivity was recovered in the neostriatal areas of animals withdrawing from morphine 24 h after [³H]lysine. Gel electrophoresis of soluble and particulate [³H]protein fractions from neostriatal tissues indicated that the gel patterns of radioactivity were not altered by chronic morphine administration. Neither morphine administration nor morphine abstinence altered the rate or amount of [³H]lysine incorporation into protein of the substantia nigra. These data demonstrate that chronic morphine administration was accompanied by a generalized increase in the amount of labelled protein transported to the neostriatum but the procedure was not sufficiently sensitive to detect a minor qualitative alteration of any particular protein(s). Furthermore, these data suggest that either the capacity or the rate of nigro-neostriatal protein transport may be increased during chronic morphine administration in the rat.     

VULNERABILITY OF THE IMMATURE RAT BRAIN TO PHENYLACETATE INTOXICATION: POSTNATAL DEVELOPMENT OF THE DETOXICATION MECHANISM

Phenylacetate is not excreted to any significant extent as the free acid in rat urine, but must be metabolized in the liver and kidney, first to phenylacetyl-CoA, then to phenylacetylglycine. One hour after [¹⁴C]phenylacetate loading, the radioactivity in the liver and kidneys of the young rat could all be accounted for as unchanged phenylacetate (50-5573, phenylacetylglycine (35–40%), and phenylacetyl-CoA (5–8%). In the brain, the radioactivity was present mainly as phenylacetate (82–90%); only 10–18% was found as phenylacetyl-CoA. The formation of phenylacetyl-CoA appeared to be the rate limiting step in the clearance of phenylacetate. In the urine at least 95% of the radioactivity was present as phenylacetylglycine, less than 1% as phenylacetate, and 3–4% as phenylacetyl-CoA. The concentration of phenylacetylglycine in the urine was therefore used as a measure of the in vivo rate of phenylacetatc clearance. This detoxication process was found to develop postnatally. The formation of phenylacetylglycine was barely detecrabie in the newborn rat and remained relatively slow for about 2 weeks. During the third week a large increase in enzymatic activity, approx 40% occurred. Adult level of activity was reached in the 40 day old rat. The extremely slow rate of detoxication in the newborn animal was reflected in the persistence of high concentrations of phenylacetate in the tissues. The relevance of our findings to human phenyl-ketonuria is discussed     

Incorporation of a kinin, N, 6-benzyladenine into soluble RNA

Kinin requiring tobacco and soybean tissues incubated on a medium containing N,6-benzyladenine-8-C¹⁴ incorporated C¹⁴ into several RNA components including adenylic and guanylic acids. About 15% of the label taken up by the tissues appeared in RNA while the remainder was distributed among several metabolites in the soluble, nonpolynucleotide fraction. Tissue grown on a kinin labeled in the side chain (N,6-benzyladenine-benzyl-C¹⁴) also incorporated a small, but nevertheless repeatable, amount of radioactivity into minor RNA components.

Ultracentrifugation studies and methylated albumin chromatography indicated that the bulk of the label from benzyladenine-benzyl-C¹⁴ is in soluble RNA. Approximately 50% of the C¹⁴ in soluble RNA is in a component which has chromatographic properties like that of benzyladenine.

It is suggested that the biological action of the kinins may hinge on their providing substituted bases in RNA in tissues which through differentiation no longer synthesize RNA-methylating enzymes. As an alternative it was hypothesized that a small amount of benzyladenine was incorporated into a m-RNA, acting there as a derepressing agent, perhaps by preventing its normal repressing function.

     

The effects of nephrectomy and probenecid on in vivo clearance of adenosine-3',5'-monophosphate from rat plasma

The disappearance of [8-³H]-adenosine 3′,5′-monophosphate (cAMP) from plasma of the intact rat has been investigated. Thirty minutes after the i.v. injection of a pulse of [³H] cAMP with 3 μmole cAMP into 300–400 g rats, more than 99% of the isotope had been removed from the plasma. The disappearance of isotope from the plasma was retarded by probenecid (20–200 mg/kg body weight), bilateral nephrectomy and bilateral nephrectomy plus hepatectomy, in increasing order of their effectiveness. Ligation of both ureters did not alter the rate of isotope disappearance. After the pulse injection, the amount of isotope in the plasma and tissues was determined and the ratio, cpm per g wet tissue/cpm per ml plasma, was calculated. Kidney cortex, kidney inner medulla and liver showed the most striking accumulations of isotope with ratios of 250, 19 and 18, respectively. Probenecid produced a dose-dependent reduction in the accumulation of isotope in kidney cortex and liver. Other tissues which showed some, albeit small, accumulation of isotope were heart (2.0), lung (2.9) and small intestine (1.6). From the accumulation of isotope in the various tissues it was estimated that the kidney cortex accounted for 39%, liver 15%, and urinary excretion 5% of the injected dose of isotope in the untreated rat. It is concluded that in the rat, at least, the kidney cortex is the principal tissue involved in cAMP removal (and degradation) from the plasma.     

Comparison of in vivo and in vitro binding of polycyclic hydrocarbons to DNA

The in vivo binding of [³H]benzo(a)pyrene (BP) and 3-[³H]methylcholanthrene (3MC) to liver and lung DNA was studied in A/J mice. Only in liver was there any reduction in total DNA-bound radioactivity between 4 h and 24 h after administration of the hydrocarbon. DNA was fractionated on Sephadex LH-20 after enzymatic digestion. A single deoxyribonucleoside-BP adduct was detected whereas two major 3MC-adducts were observed. With both BP and 3MC, three additional peaks of radioactivity eluted rapidly in the lung DNA experiments while a fourth was noted with liver DNA. The nucleoside-bound adducts from lung represented a much larger proportion of the total radioactivity than with liver. In vitro analysis of 3MC binding to DNA showed the nucleoside-bound adducts to be predominantly deoxyguanosine-dependent but that the early peaks were independent of base suggesting binding to another part of the DNA molecule, perhaps phosphate, i.e., phosphotriesters.     

Deacetylation of N8-acetylspermidine by subcellular fractions of rat tissue

The in vitro deacetylation of N⁸-acetylspermidine by an enzyme activity in rat tissues is described. This deacetylase activity occurs as a soluble, cytoplasmic enzyme in rat liver and was detected in the 100,000g supernatant fraction of all tissues examined. The highest specific activity was found in liver. Spleen, kidney, and lung were found to contain 20–50% of the activity in liver, while heart, brain, and skeletal muscle exhibited from 2 to 10% of the activity in liver. Serum contained only barely detectable levels of activity, much lower than any of the tissues studied. The in vitro metabolism of N¹-acetylspermidine differed from that observed for N⁸-acetylspermidine and does not appear to involve a simple deacetylation reaction.     

Characterization by high performance liquid chromatography of nuclear metabolites of testosterone in the brains of male rhesus monkeys

Testosterone (T) restores the potency of castrated male rhesus monkeys, and our autoradiographic data have demonstrated that ³H-T or its metabolites concentrate in cell nuclei in the corticomedial amygdala, bed nucleus of stria terminalis, preoptic area, and hypothalamus. In rat, ³H-estradiol (³H-E₂) is a major nuclear metabolite of ³H-T in areas of the limbic system, but comparable data are lacking for the primate. We have therefore developed an improved technique using high performance liquid chromatography for investigating metabolites of ³H-T that accumulate in cell nuclei in small amounts of tissue obtained from the brain of the rhesus monkey. Two castrated male rhesus monkeys were injected with 5 mCi of ³H-T and were killed 30 min later. In amygdala, preoptic area-bed nucleus of stria terminalis, and hypothalamus, 48–70% of the nuclear radioactivity was in the form of ³H-E₂ (Type I tissues). In six other brain areas and in pituitary, 35–85% of the nuclear radioactivity was in the form of ³H-T (Type II tissues), whereas in genital tract tissues, 86–99% of the nuclear radioactivity was in the form of ³H-dihydrotestosterone (³H-DHT) (Type III tissues). In plasma and in supernatants from both Type I and Type II tissues, the proportions of ³H-T were high, and ³H-E₂ did not exceed 10% of the total extractable radioactivity. These data suggest that, as in rodents, some of the central actions of T in primates may be mediated by estrogen target neurons.