Design, synthesis, and evaluation of cyclofenil derivatives for potential SPECT imaging agents

To develop technetium- and rhenium-labeled nonsteroidal estrogen imaging agents for estrogen receptor (ER) positive breast tumors, two groups of rhenium and technetium cyclofenil derivatives were synthesized and characterized. The binding affinities of the rhenium complexes for ERs were determined. The tricarbonyl rhenium complex showed the highest binding affinity for ERs (81.2 for ERβ, 16.5 for ERα). Tricarbonyl technetium cyclofenil complexes were obtained in high radiochemical purity and radiochemical yields. The results of studies of their octanol/water partition and in vitro stability are presented. These results demonstrate that these radiolabeled cyclofenil derivatives may be considered as potential breast cancer imaging agents.     

Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen.

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.     

Treatment of primary hyperparathyroidism with cyclofenil--a synthetic stilbestrol derivative with minimal feminizing effects

In eight patients with primary hyperparathyroidism (HPT) the laboratory effects of cyclofenil, which is a synthetic stilbestrol derivative with weak feminizing effects, were studied over a period of 5-13 weeks. In all the patients there were during treatment clear reductions of the serum calcium levels as well as the urinary excretions of calcium and hydroxyproline. These findings are in accordance with earlier reports that oestrogens reduce bone resorption mediated by parathyroid hormone. It has previously been demonstrated that cyclofenil can be given, on other indications, for several years to both male and female patients without oestrogenic side-effects. This pilot study, therefore, indicates that treatment with cyclofenil might be of value in some cases of primary HPT where surgery is not considered.     

Design and synthesis of functionalized cyclopentadienyl tricarbonylmetal complexes for technetium-94m PET imaging of estrogen receptors

Cyclopentadienyl tricarbonylmetal (CpTM, M = Re, (94m)Tc) complexes, some based on a typical nonsteroidal estrogen, were prepared with the aim of developing technetium- and rhenium-labeled imaging agents for estrogen receptor (ER) positive breast tumors. CpT[(94m)Tc] compounds with simple cyclopentadienyl substituents were first synthesized using a modified double ligand transfer reaction. The in vivo biodistribution of one of these CpT[(94m)Tc] complexes was determined by tissue dissection and microPET imaging. Novel C-ring substituted analogues of cyclofenil, a nonsteroidal compound known to bind the ER, were also prepared, and their ER binding was measured. Because of their low ER affinity, however, labeling and imaging studies of these compounds were not pursued. It is notable that the highest ER binding analogue, a CpTRe cyclofenil derivative, could be synthesized from the corresponding ferrocenyl cyclofenil analogue by the double ligand transfer reaction. This further demonstrates the versatility of the double ligand transfer reaction and indicates that the synthesis of technetium and rhenium radiolabeled agents for breast tumor imaging and therapy is also likely to be successful.     

Adrenal versus nonadrenal sympathetic preganglionic neurones in the lower thoracic intermediolateral nucleus of the cat: physiological properties

Adrenal and nonadrenal sympathetic preganglionic neurones (SPNs) in the intermediolateral nucleus of spinal segments T8-T10 in the cat were compared according to a number of physiological properties. An SPN was classified as "adrenal" (n = 37) if it could be antidromically activated by electrical stimulation of the adrenal medulla. An SPN that could not be activated from the adrenal medulla yet could be antidromically activated by electrical stimulation of the greater splanchnic nerve was classified as "nonadrenal" (n = 123). Approximately 50% of adrenal SPNs (17 out of 37) were activated antidromically by stimulation of both the greater splanchnic nerve and adrenal medulla, suggesting that these neurones projected to the adrenal medulla via the greater splanchnic nerve, with the other adrenal SPNs taking a different route. The mean conduction velocities of adrenal (6.7 +/- 1.8 (SD) m/s) and nonadrenal (6.7 +/- 1.5 m/s) sympathetic preganglionic axons were similar. Over 80% of adrenal (31 out of 37) and nonadrenal (104 out of 116) SPNs were spontaneously active. The two types of neurone were indistinguishable in terms of the rates and patterns of discharge. Adrenal SPNs discharged with a mean rate of 1.4 +/- 1.1 spikes/s, and nonadrenal SPNs discharged with a mean rate of 1.8 +/- 1.4 spikes/s. With both types of SPN, the pattern of spontaneous activity was either irregular or phasic. With the latter pattern, periodic bursts of discharge were at the same frequency as oscillations in arterial pressure, frequency of ventilation, or phrenic nerve discharge. These data suggest that adrenal and nonadrenal sympathetic preganglionic neurones in the intermediolateral nucleus in caudal thoracic segments share a number of common physiological properties.     

Influences of the adrenal gland and photoperiod on the hamster oestrus cycle

The influence of the adrenal gland and photoperiod on the adult female hamster estrous cycle was investigated. Hamsters were exposed to 14 hours of light per day and estrous cycles were monitored. Normal cycles were characterized by a copious discharge every 4th day. Hamsters were then adrenalectomized, adrenalectomized and blinded, or blinded only. Adrenalectomized/blinded hamsters were given a .2% sodium saccharine-1%(w/v) sodium chloride solution to drink which promotes long-term survival in most adrenalectomized hamsters. With blinding alone, it took from 18 to 62 days for cessation of estrous cycles. Cycles ceased in a mean of 36.2 days in blinded/adrenalectomized hamsters. After adrenalectomy only, 3 hamsters continued to show 4-day estrous cycles for at least 9 weeks in response to the long days. 6 adrenalectomized animals died due to adrenal insufficiency but displayed several 4-day estrous cycles. It is suggested that in the hamster, adrenal hormones are nonessential for the long-term expression of estrous cyclicity. However, further research is needed to determine whether adrenal hormones are capable of modulating photoperiodic time measurement or ovarian regression.     

Biochemical Diagnosis of Adrenal Insufficiency: the Added Value of Dehydroepiandrosterone Sulfate Measurements

ObjectiveTo review biochemical tests used in establishing the challenging diagnosis of adrenal insufficiency.MethodsWe reviewed the relevant literature, including our own data, on various biochemical tests used to determine adrenal function. The advantages and limitations of each approach are discussed.ResultsBaseline measurements of serum cortisol are helpful only when they are very low (≤ 5 μg/dL) or clearly elevated, whereas baseline plasma adrenocorticotropic hormone levels are helpful only when primary adrenal insufficiency is suspected. Measurements of baseline serum dehydroepiandrosterone sulfate (DHEA-S) levels are valuable in patients suspected of having adrenal insufficiency. Although serum DHEA-S levels are low in patients with primary or central adrenal insufficiency, a low level of this steroid is not sufficient by itself for establishing the diagnosis. A normal age- and sex-adjusted serum DHEA-S level, however, practically rules out the diagnosis of adrenal insufficiency. Many patients require dynamic biochemical studies, such as the 1-μg cosyntropin test, to assess adrenal function.ConclusionIn establishing the diagnosis of central adrenal insufficiency, we recommend measurements of baseline serum cortisol and DHEA-S levels. In addition to these, determination of plasma levels of aldosterone, adrenocorticotropic hormone, and renin activity is necessary when primary adrenal insufficiency is suspected. With a random serum cortisol level of ≥ 12 μg/dL in the ambulatory setting or a normal age- and sex-adjusted DHEA-S level (or both), the diagnosis of adrenal insufficiency is extremely unlikely. When serum DHEA-S levels are low or equivocal, however, dynamic testing will be necessary to determine hypothalamic-pituitary-adrenal axis function. (Endocr Pract. 2011;17:261-270)     

Effects of atrial natriuretic peptide AP II on the morphofunctional state of the adrenal cortex of white rats]

The effect of atrial natriuretic peptides synthetic analog AP II on adrenal zona glomerulosa and zona fasciculata physiological regeneration have been studied on male rats. The 3H-thymidine incorporation into DNA in adrenal cortical cells was evaluated in 4 and 24 h after intraperitoneal injection of 10 or 100 mcg/kg AP II. Besides, we have investigated the influence of AP II on adrenal cortical cells karyometric parameter in 4 and 24 h and aldosterone plasma concentration in 1 h after injection. 10 mcg/kg AP II increased the fraction of labelled nuclei in zona glomerulosa and decreased the aldosterone plasma level. No significant changes were seen in zona fasciculata cells proliferation. 100 mcg/kg AP II inhibited the DNA synthesis process in adrenal zona fasciculata, but had no significant influence on zona glomerulosa physiological regeneration and aldosterone plasma concentration. No nuclear morphometric parameter changes were observed in adrenal glomerulosa and fasciculata cells of AP II--treated animals.     

A premature increase in circulating cortisol suppresses expression of 11beta hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid in the adrenal of the fetal sheep

We have investigated the effect of intrafetal cortisol administration, before the normal prepartum cortisol surge, on the expression of 11beta hydroxysteroid dehydrogenase (11betaHSD) type 2 mRNA in the fetal adrenal. We also determined whether increased fetal cortisol concentrations can stimulate growth of the fetal adrenal gland or increase expression of adrenal steroidogenic enzymes. Cortisol (hydrocortisone succinate: 2.0-3.0 mg in 4.4 ml/24 h) was infused into fetal sheep between 109 and 116 days of gestation (cortisol infused; n = 12), and saline was administered to control fetuses (saline infused; n = 13) at the same age. There was no effect of cortisol infusion on the fetal adrenal:body weight ratio (cortisol: 101.7 +/- 5.3 mg/kg; saline: 108.2 +/- 4.3 mg/kg). The ratio of adrenal 11betaHSD-2 mRNA to 18S rRNA expression was significantly lower, however, in the cortisol-infused group (0.75 +/- 0.02) compared with the group receiving saline (1.65 +/- 0.14). There was no significant effect of intrafetal cortisol on the relative abundance of adrenal CYP11A1, CYP17, CYP21A1, and 3betaHSD mRNA. A premature elevation in fetal cortisol therefore resulted in a suppression of adrenal 11betaHSD-2. Increased intra-adrenal exposure to cortisol at this stage of gestation is, however, not sufficient to promote adrenal growth or steroidogenic enzyme gene expression.     

Stress-protective effect of the synthetic ACTH-like peptide leucocorticotropin

We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81-88 sequence of the precursor of human interleukin-1alpha ([3H]GKVLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (Kd 2.2 +/- 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10-20 microg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 microg/animal, three times) or significantly decreases (at a dose of 10 microg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor.     

Inhibition of the stimulatory effect of ACTH on adrenal and ovarian blood flow by indomethacin in the dog.

In anoestrous dogs heated thermocouples were inserted into one of the adrenals and ovaries. The basal adrenal and ovarian blood flow was recorded and enhanced by 1.2, 2.5 or 5.0 micrograms/kg of intravenous ACTH before and after the intravenous administration of 20 or 50 mg/kg of indomethacin. Indomethacin, depending on the dose and on the time elapsing after its administration inhibited the enhancing effect of ACTH on adrenal and ovarian blood flow. Since indomethacin is a specific inhibitor of prostaglandin synthesis it has been suggested that prostaglandins act as mediators in the vasodilatation elicited by ACTH in the canine adrenal and ovary.     

Effects of microwaves on the adrenal cortex

Six-hundred-and-one male Long-Evans rats were used to study the effect of microwaves on adrenocortical secretion. Power density ranged from 0.1 to 55 mW/cm2 (SAR 0.02 to 11 W/kg). The microwave signal was 2.45 GHz amplitude modulated at 120 Hz. Serum corticosterone (CS) concentration was used as an index of adrenocortical function. Ten different exposure protocols were used to identify confounding factors influencing the sensitivity of adrenal cortex to microwave exposure. Increases in CS concentration were proportional to power density or colonic temperature and inversely proportional to the baseline CS. Increased CS concentration was never observed without increased colonic temperature and was not persistent 24 h after exposure. Acclimation (reduction in magnitude of response) could be noted after the tenth exposure. Facilitated heat loss attenuated the magnitude of CS increases by limiting the degree of hyperthermia. Ethanol enhanced the hyperthermic response and desensitized the adrenal response to microwave hyperthermia by increased baseline CS. Ether stimulated adrenal secretion irrespective of previous microwave exposure or adrenal stimulation induced by microwaves. Minor inhibition was also noted occasionally as decreased CS concentration at lower intensity (less than 20 mW/cm2) and decreased postexposure urinary CS excretion at 40 mW/cm2. Adrenal stimulation required minimally a 20 mW/cm2 (4 W/kg) or 0.7 degrees C increase in colonic temperature. An SAR lower than 4 W/kg may stimulate adrenal secretion by potentiating the hyperthermic effect if the ambient temperature is well above 24 degrees C.     

Comparison of the effects of neurotensin and ACTH on the pituitary-adrenocortical axis of dexamethasone-suppressed rats.

Neurotensin (NT) (12-48 micrograms/kg-1/day-1, for 2 days, s.c.), like ACTH (60 micrograms/kg-1/day-1, for 2 days, s.c.), counteracted the dexamethasone (Dx)-induced (120 micrograms/kg-1/day-1, for 4 days, s.c.) adrenal zona-fasciculata cell atrophy. NT notably raised, in Dx-suppressed rats, the plasma concentration of ACTH, which reached about that found after exogenous ACTH administration. However, at variance with ACTH, NT did not enhance either plasma corticosterone (B) level or B production by adrenal quarters in vitro. The conclusion is drawn that NT modulates the function of the rat pituitary-adrenocortical axis, by simultaneously stimulating hypophyseal ACTH release and inhibiting steroidogenesis at the adrenal level.     

IGF1 and 2 in two models of adrenal growth

Insulin-like growth factors (IGFs) 1 and 2 were measured in the adrenal glands of rats undergoing either compensatory growth following left unilateral adrenalectomy or adrenal regeneration following bilateral adrenal enucleation. In normal rat adrenal gland, the tissue concentration of IGF2 (7.45 +/- 0.99 pg/micrograms protein) wa higher than IGF1 (1.26 +/- 0.23 pg/micrograms protein), both peptides being more abundant in the inner zones of the adrenal gland compared to the capsule-glomerulosa. During compensatory growth of the right adrenal gland, IGF1 and 2 increased significantly compared with control right adrenal glands at 24 h following left unilateral adrenalectomy (P less than 0.001). At 68 h, the increase remained significant for IGF1 (P = 0.012). The two peptides were measured in the regenerating adrenal gland at 7, 14 and 21 days following bilateral enucleation. Whilst there was a trend towards an increase in the IGF1 and 2 content of regenerating adrenal glands, the increase was significant only for IGF2 in the left adrenal gland at 21 days following enucleation. Plasma IGF1 and 2 did not increase compared to controls during the experiments (110.97 +/- 1.95 and 46.33 ng/ml, respectively), suggesting that the changes in tissue IGF reflect increased local production during rapid growth of the adrenal gland.     

Stress-protective effect of the synthetic ACTH-like peptide leucocorticotropin

We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81–88 sequence of the precursor of human interleukin-1α ([³H]GK VLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (K _d 2.2 ± 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10–20 μg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 μg/animal, three times) or significantly decreases (at a dose of 10 μg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor.     

Gonadal steroids modulate adrenal fasciculata 3 beta-hydroxysteroid dehydrogenase-isomerase activity in mice.

The observation that adrenal 3 beta-hydroxysteroid dehydrogenase-delta 5----delta 4-isomerase (3 beta HSD) activity is greater in females than in males of both C57BL/6J and C3H/HeJ inbred strains of mice led us to test the hypothesis that this enzyme's activity within the adrenal gland may be modulated by gonadal steroids. Two weeks after sham-operation or castration, no castration effect was seen in adrenal 3 beta HSD activity, but the sex difference had been eliminated in C57BL/6J animals. Gonadal steroids were replaced in castrated animals of both sexes of C57BL/6J and C3H/HeJ mice. Daily injections of androgenic steroids (testosterone propionate or dihydrotestosterone) decreased adrenal 3 beta HSD activity in both sexes of both strains of mouse. Although estradiol benzoate did not alter adrenal 3 beta HSD activity in either sex or strain tested when the activity was expressed on a per adrenal gland basis, it did inhibit adrenal 3 beta HSD activity when expressed on a per milligram of adrenal tissue basis in C57BL/6J but not in C3H/HeJ animals. Histochemical staining for 3 beta HSD and the relationship between adrenal weight and the cross-sectional area of the zona fasciculata suggested that the adrenal zona fasciculata was the target of the inhibitory effects of androgens on adrenal 3 beta HSD activity.     

Lack of an adrenal cortex in Sf1 mutant mice is compatible with the generation and differentiation of chromaffin cells

The diversification of neural-crest-derived sympathoadrenal (SA) progenitor cells into sympathetic neurons and neuroendocrine adrenal chromaffin cells was thought to be largely understood. In-vitro studies with isolated SA progenitor cells had suggested that chromaffin cell differentiation depends crucially on glucocorticoids provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor gene had revealed that adrenal chromaffin cells develop mostly normally in these mice. Alternative cues from the adrenal cortex that may promote chromaffin cell determination and differentiation have not been identified. We therefore investigated whether the chromaffin cell phenotype can develop in the absence of an adrenal cortex, using mice deficient for the nuclear orphan receptor steroidogenic factor-1 (SF1), which lack adrenal cortical cells and gonads. We show that in Sf1-/- mice typical chromaffin cells assemble correctly in the suprarenal region adjacent to the suprarenal sympathetic ganglion. The cells display most features of chromaffin cells, including the typical large chromaffin granules. Sf1-/- chromaffin cells are numerically reduced by about 50% compared with the wild type at embryonic day (E) 13.5 and E17.5. This phenotype is not accounted for by reduced survival or cell proliferation beyond E12.5. However, already at E12.5 the 'adrenal' region in Sf1-/- mice is occupied by fewer PHOX2B+ and TH+ SA cells as well as SOX10+ neural crest cells. Our results suggest that cortical cues are not essential for determining chromaffin cell fate, but may be required for proper migration of SA progenitors to and/or colonization of the adrenal anlage.     

Studies on the effects of ethinyl estradiol and norethisterone acetate on the adrenal cortex and some other tissues in the rat.

Alkaline phosphatase activity (AP) of the adrenal cortex of rats were determined under the effect of ethinyl estradiol (EE) and/or norethisterone acetate (NA), the two components of the contraceptive pill gyn-anovlar (Schering AG Berlin). A pathological study was also carried out to examine the effects of EE and NA on other tissues mainly the liver, lungs, spleen and ovaries. EE in a dose of 10 micrograms/day for 2 weeks caused a significant increase in the weight of the adrenal but no significant increase in the AP/g cortical tissue. The 25 and 50 micrograms doses for the same period caused a significant increase in both adrenal weight and AP. When treatment was prolonged to 6 weeks no effect on adrenal weight or AP was detected. The same finding was obtained with NA in a dose of 7 mg/rat/day for 2 weeks. The 14 mg dose of NA for the same period caused a significant increase in adrenal weight but no effect on AP. The 21 mg dose caused a significant increase in adrenal weight accompanied by significant decrease in AP/g cortical tissue. Treatment with NA for 6 weeks caused a rise in adrenal weight and AP with the 7 mg dose, then a decline in AP with the 14 mg dose, and a decline in both adrenal weight and AP with the 21 mg dose. As regards the effects of EE and NA on other tissues, EE was found to have a powerful stimulatory effect on the reticuloendothelial system (RES) as well as toxic effects on the liver. NA did not produce such lesions except for the large doses and prolonged periods of treatment. In addition NA produced cholestasis in the liver associated with staining of the liver cells with bile. Combination of EE and NA in the form of gyn-anovlar produced more powerful stimulation of RES and decreased the toxic manifestations of either component. As regards the ovaries, administration of 50 micrograms EE for 6 weeks produced profound hyperplasia of the granulosa cells of the Graafian follicles and inhibited ovulation, however, NA did not inhibit ovulation. With gyn-anovlar, the effect of EE on the ovaries seemed to predominate and ovulation was inhibited.     

Rates of cholesterol synthesis and low-density lipoprotein uptake in the adrenal glands of the rat, hamster and rabbit in vivo

The absolute rate of cholesterol acquisition from de novo synthesis and from receptor-dependent and receptor-independent low-density lipoprotein (LDL) uptake was determined in the adrenal glands of the rat, hamster and rabbit under in vivo conditions. The rate of incorporation of [3H]water into cholesterol in the adrenal gland was much higher in the hamster (1727 nmol/h per g) and rabbit (853 nmol/h per g) than in the rat (71 nmol/h per g). Assuming that 23 atoms of 3H are incorporated into the cholesterol molecule during its biosynthesis, the absolute rates of cholesterol synthesis were then calculated to equal 59, 29 and 2.4 micrograms/h per g of adrenal gland in the hamster, rabbit and rat, respectively. Rates of LDL-cholesterol uptake were measured using a primed continuous infusion of [14C]sucrose-labeled homologous LDL (total LDL transport) and methylated human LDL (receptor-independent LDL transport). The rate of total LDL-cholesterol uptake in the adrenal gland was much higher in the rabbit (227 micrograms/h per g) than in the rat (18 micrograms/h per g) or hamster (6 micrograms/h per g). In all three species LDL uptake was mediated largely (greater than 93%) by receptor-dependent mechanisms. In terms of total cholesterol acquisition, the hamster adrenal gland derived 10-times more cholesterol from de novo synthesis than from LDL uptake, whereas the converse was true in the rabbit. Rates of de novo synthesis and LDL-cholesterol uptake were both low in the rat adrenal gland, which is known to derive cholesterol mainly from circulating high-density lipoproteins. Thus, the adrenal gland acquires cholesterol for hormone synthesis from at least three different sources and the quantitative importance of these sources varies markedly in different animal species, including man.