Retention volume of some steroids during their separation by high performance liquid chromatography

The volume of retention of corticosterone, hydrocortisone, aldosterone, dexamethasone, cortisone, methylprednisolone, and prednisolone during their separation by high-performance liquid chromatography was evaluated. The studies were performed on a Milichrome microcolumn chromatographer. The order of glucocorticoid release and volume of their retention were under study. Measurement of endogenous and exogenous plasma glucocorticoids is practically important in clinical medicine. Steroid concentrations indicate the pharmacodynamics and pharmacokinetics of exogenous glucocorticoids and their effects on the production of endogenous steroids and reflect the biorhythms of corticosterone and hydrocortisone.     

GLUCOCORTICOIDS AND CELLULAR IMMUNITY IN VITRO : FACILITATION OF THE SENSITIZATION PHASE AND INHIBITION OF THE EFFECTOR PHASE OF A LYMPHOCYTE ANTI-FIBROBLAST REACTION

We studied the influence of glucocorticoids on the sensitization phase as well as on the cytolytic effector phase of an in vitro lymphocyte-mediated immune reaction. Lymphocytes obtained from the spleens or lymph nodes of unimmunized inbred rats were sensitized against foreign rat or mouse embryonic fibroblasts in cell culture. The capacity of the sensitized lymphocytes to produce a cytolytic effect was tested by transferring them to target fibroblast cultures. Injury to target fibroblasts was measured by release of radioactive ⁵¹Cr from previously labeled fibroblasts or by direct count of viable fibroblasts after incubation with sensitized lymphocytes. Various concentrations of water-soluble hydrocortisone or prednisolone were added to cell cultures during the 5 day sensitization phase and/or during the subsequent cytolytic effector phase and the influence of these hormones on the number and cytolytic capacity of the lymphocytes was measured. During the sensitization phase, the presence of glucocorticoid hormones, at concentrations of about 1 µg/ml, led to a profound decrease in the total number of recoverable lymphocytes. However, the per cent of large transformed lymphocytes was much greater in these treated cultures. The antigen-specific cytolytic capacity per cell of the glucocorticoid-treated lymphocytes, after the hormone was removed, was several times greater than that of lymphocytes sensitized in the absence of added hormones. Glucocorticoids influenced the effector phase of the reaction by inhibiting lymphocyte-mediated injury to target fibroblasts. The hormones, at concentrations of about 1 µg/ml, inhibited the cytolytic effect by about 50% without reducing the viability of the sensitized lymphocytes. Dose-dependent toxicity to lymphocytes and increasing inhibition of cytolytic effect appeared at higher concentrations of hormones. Thus, hydrocortisone and prednisolone, at concentrations of about 1 µg/ml, did not suppress the induction of sensitization, a process which they seem to facilitate in vitro. However, similar concentrations of these hormones appear to inhibit the cytolytic effector mechanism of sensitized lymphocytes. These findings may be relevant to the use of glucocorticoids as immunosuppressive agents in vivo.     

应用高效液相色谱-二极管阵列检测器筛查中药及保健品中糖皮质激素类非法添加剂

目的探讨应用高效液相色谱-二极管阵列检测器(HPLC-DAD)技术建立中药及保健品中非法添加氢化可的松琥珀酸钠、泼尼松龙、氢化可的松、泼尼松、甲泼尼龙、倍他米松、地塞米松、曲安奈德、醋酸泼尼松龙、醋酸氢化可的松、醋酸氟氢可的松、醋酸泼尼松、醋酸可的松、醋酸地塞米松、丁酸氢化可的松、醋酸曲安奈德、醋酸氟轻松和哈西奈德等18种糖皮质激素类添加剂成分的检查方法。方法采用Alltima C18色谱柱(4.6 mm×250 mm,5μm),流动相为乙腈-10 mmol/L醋酸铵二元梯度洗脱,流速为0.5 m L/min,检测波长242nm。对于HPLC-DAD检出的疑似阳性样品用液相色谱-质谱法(LC-MS/MS)进行进一步确认。结果氢化可的松和泼尼松的色谱峰重叠,其他16种糖皮质激素在5~320 mg/L内线性关系良好,线性相关系数(r)均大于0.9998;灵敏度高,最低检测限均在0.49~0.51 ng之间,定量限均在1.23~1.27 ng之间;回收率良好,低、中、高3个浓度的回收率均在97.52%~104.2%之间;精密度良好,相对标准偏差(RSD)均小于0.47%;稳定性良好,12 h内峰面积和保留时间的相对标准偏差(RSD)均小于0.68%。14种不同剂型的样品通过HPLC-DAD检查和LC-MS/MS确认,结果显示:某片剂中检测出醋酸地塞米松,某喷雾剂中检出倍他米松。结论 HPLC-DAD技术应用快速、灵敏、易推广,可作为筛查药品及保健品中非法添加18种糖皮质激素的检查方法。     

Inhibition of immunoglobulin, but not polypeptide base-stimulated release of histamine and arachidonic acid by anti-inflammatory steroids

Incubation overnight of purified rat mast cells with glucocorticoids inhibited the release of histamine and [1-14C]arachidonic acid (and its metabolites) stimulated by three immunoglobulin (Ig) E-like secretagogues, anti-IgE, the antigen-ovalbumin and concanavalin A. In contrast, pretreatment with glucocorticoids did not affect either histamine or [1-14C]arachidonic acid release stimulated by somatostatin, compound 48/80 or the calcium ionophore A23187. Glucocorticoids inhibited IgE-like arachidonic acid and histamine release with an order of potency similar to their in vivo anti-inflammatory potencies (i.e., fluocinolone greater than dexamethasone greater than hydrocortisone greater than cortisone). This inhibition required several hours and was temperature-dependent, suggesting a specific glucocorticoid receptor mechanism. IgE-stimulated Ca++ influx was decreased by hydrocortisone pretreatment. These results suggest that glucocorticoids specifically uncouple IgE-mediated calcium flux with subsequent inhibition of histamine and arachidonic acid release.     

Biochemical actions of glucocorticoids on macrophages in culture. Specific inhibition of elastase, collagenase, and plasminogen activator secretion and effects on other metabolic functions

The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased by 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased by 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1--1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contrast, secretion of lysozyme was not affected by glucocorticoids or other steroids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Effects were detected within 1--6 h after addition of the glucocorticoids, became maximum by 24 h, and were reversed during a similar time period after removal of the hormones. The extent of inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1--10 nM) similar to those that half-saturated the specific glucocorticoid receptors in these cells. At high concentrations of dexamethasone (100--1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (greater than 95%) than the secretion of elastase (60--80%). Progesterone alone had no effect on secretion, but it blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1--100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone. These data support the regulatory role of glucocorticoids on macrophage functions at physiological concentrations.     

Eosinophil hematopoietins antagonize the programmed cell death of eosinophils. Cytokine and glucocorticoid effects on eosinophils maintained by endothelial cell-conditioned medium.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was established as the constitutive and elicited human umbilical vein endothelial cell-derived eosinophil viability-sustaining factor. Stimulation of endothelium cell monolayers with IL-1 alpha (5 U/ml) increased the 48-h elaboration of GM-CSF from a mean of 3.2 to a mean of 8.2 pM (P less than 0.05). Dexamethasone (100 nM) decreased the constitutive GM-CSF elaboration by 49% (P less than 0.001) but did not diminish production by IL-1 alpha-stimulated endothelium. However, eosinophil viability decreased by 21% in dexamethasone-pretreated IL-1 alpha-stimulated endothelial cell-conditioned medium (P less than 0.05), which suggested viability antagonism by glucocorticoids. After 24 h of culture, eosinophil viability for replicate cells in enriched medium alone or with 1 pM GM-CSF decreased from means of 43 and 75% to means of 21 and 54%, respectively, when dexamethasone was included (P less than 0.05). However, 10 pM GM-CSF, IL-3, or IL-5 protected the cells against dexamethasone and against endonuclease-specific DNA fragmentation. In this model system of eosinophil-tissue interactions, dexamethasone prevents the endothelial cells from inducing a pathobiologic phenotypic change in the eosinophil by suppression of GM-CSF elaboration to concentrations that are not cytoprotective. Cytokine priming by GM-CSF, IL-3, or IL-5 may account for the differential responsiveness of select eosinophilic disorders to glucocorticoids.     

Effect of dexamethasone on reactive oxygen species generation by leukocytes and plasma interleukin-10 concentrations: a pharmacodynamic study.

After the demonstration that hydrocortisone inhibits reactive oxygen species (ROS) generation by leukocytes in vivo in a highly predictable manner, we investigated the effect of dexamethasone at a dose of 4 mg, which is thought to be roughly equivalent to 100 mg hydrocortisone. We also tested the hypothesis that dexamethasone may increase the plasma concentration of interleukin-10 (IL-10), an immunomodulatory cytokine that inhibits T(H)1 cells. Dexamethasone (4 mg given intravenously) markedly inhibited ROS generation by mononuclear cells and polymorphonuclear leukocytes. The onset of the effect on polymorphonuclear leukocytes occurred at 1 hour (76.3% +/- 9.3% of basal value), and the peak effect occurred at 4 hours (22.9% +/- 6.4% of basal value), with a significant inhibition still persistent at 8 hours (51.3% +/- 14.3% of basal value; F = 66.7; P < .001). ROS generation was restored to baseline at 24 hours (97.6% +/- 9.5%). The inhibitory effect of dexamethasone on mononuclear cells was 78.3% +/- 9.5% of baseline at 1 hour, 11.4% +/- 6.6% at 4 hours, 30.3% +/- 14.1% at 8 hours, and 102.3% +/- 18% at 24 hours (F = 66.5; P < .001). The peak inhibitory effect of dexamethasone on mononuclear cells (11.4% +/- 6.6%) was significantly greater (P < .05) than that on polymorphonuclear leukocytes (22.9% +/- 6.4%). Plasma IL-10 concentrations increased consistently from 4.8 +/- 1.8 pg/mL within 1 hour of dexamethasone injection and peaked at 4 hours (8.8 +/- 2.3 pg/mL), declining to baseline at 8 hours (F = 4.26; P < .004). Dexamethasone (and possibly other glucocorticoids) therefore exerts its immunosuppressive and anti-inflammatory effects by inhibiting ROS generation by leukocytes and by increasing the plasma concentrations of IL-10.     

糖皮质激素对于活化RBL-2H3表达IL-5的影响

目的:探讨糖皮质激素对于活化RBL-2H3(简称RBL)表达白细胞介素5(IL-5)的影响。方法:利用抗原和钙离子载体A23187分别激活RBL表达IL-5,通过总RNA提取、逆转录和半定量PCR研究加入糖皮质激素后对于RBL活化表达IL-5的影响。结果:经A23187活化的RBL,加入10-7mol/L地塞米松后,其IL-5表达量降低到对照的9%,加入10-6mol/L氢化可的松后,其IL-5表达量降低到对照的8.5%。经抗原活化的RBL,加入10-6mol/L地塞米松后,其IL-5表达量降低到对照的12%,加入10-6mol/L氢化可的松后,其IL-5表达量降低到对照的9%。结论:糖皮质激素对于活化RBL表达IL-5具有极大的抑制作用,提示糖皮质激素在临床上治疗哮喘的作用与其抑制肥大细胞表达IL-5存在一定关系。     

Cell adhesion molecules as a marker reflecting the reduction of endothelial activation induced by glucocorticoids

In vitro, steroids down-regulate the expression of cell adhesion molecules (CAMs) in endothelial cells stimulated by lipopolysaccharide. Low-dose hydrocortisone is a new treatment of patients with septic shock, a state that is characterized by an endothelial injury. The aim of the present study was to investigate whether the plasma levels of soluble CAMs, reflecting in vivo endothelial activation, could be modulated in patients with septic shock treated by hydrocortisone. This was a prospective and observational study conducted in the intensive care unit at a university hospital. The subjects included 40 patients with septic shock (American College of Chest Physicians Consensus Conference/Society of Critical Care Medicine definition); 45 healthy blood donors served as controls. The patients receiving the standard care ("reference group") during the first 6 months were compared with the patients receiving the hydrocortisone therapy ("hydrocortisone group") for the next 6 months. Measurements of sCAMs were performed on days 1 and 3 of the disease. On day 1, sE-selectin, sP-selectin, sVCAM-1, and sICAM-1 were significantly elevated in patients with septic shock compared with healthy donors. sE-selectin levels significantly decreased between days 1 and 3 in the "hydrocortisone group," whereas there was no significant change in the "reference group". Surprisingly, sICAM-1 levels significantly increased between days 1 and 3 only in patients treated by hydrocortisone. No significant changes were observed for sP-selectin and sVCAM-1 levels in the two groups. In patients with septic shock, glucocorticoids differently affected the pattern of evolution of sCAMs, with sE-selectin being decreased and sICAM-1 being increased. Expression of sP-selectin and sVCAM-1 was not affected.     

GLUCOCORTICOID SUPPRESSION OF MACROPHAGE MIGRATION INHIBITORY FACTOR

The ability of hydrocortisone to modify antigen-mediated inhibition of macrophage migration, an in vitro correlate of cellular immunity in the guinea pig, was investigated. Only the glucocorticoids, hydrocortisone and dexamethasone, significantly blocked migration inhibitory factor (MIF) activity in pharmacologic concentrations. Hydrocortisone had no effect on antigen "processing" by macrophages, nor on the ability of antigen-stimulated peritoneal exudate lymphocytes to produce MIF. Rather, hydrocortisone antagonized directly the inhibitory effect of MIF on the macrophage.     

Role of antioxidant defenses against ethanol-induced damage in cultured rat gastric epithelial cells

Reactive oxygen species appears to be involved in the pathogenesis of ethanol-induced gastric mucosal injury in vivo. Because ingested ethanol diffuses into the gastric mucosa, targeting both epithelium and endothelium, in the present study we examined the possible protective effect of antioxidants on ethanol damage in gastric epithelial cells and endothelial cells in vitro. Cytotoxicity by ethanol was quantified by measuring 51Cr release. The effects of impairment of the glutathione redox cycle and of inhibition of cellular catalase were examined. The generation of superoxide was assessed by the reduction in cytochrome c. Ethanol caused a time- and dose-dependent increase in 51Cr release from epithelial cells. Incubation of cells with DL-buthionine-(S,R)-sulfoximine, while reducing glutathione production, dose dependently enhanced ethanol-induced injury. 1,3-Bis(chloroethyl)-nitrosourea, while inhibiting glutathione reductase activity, also sensitized cells to ethanol. In contrast, the inhibition of catalase with 3-amino-1,2, 4-triazole did not alter the susceptibility of epithelial cells to ethanol. Ethanol induced damage to endothelial cells in a similar fashion. In endothelial cells, however, neither impairment of the glutathione cycle nor inhibition of catalase influenced ethanol-induced damage. Epithelial cells, when exposed to ethanol, increased superoxide production as a function of ethanol concentration, whereas endothelial cells did not. The glutathione redox cycle, but not cellular catalase, plays a critical role in protecting epithelial cells against ethanol damage, whereas neither antioxidant seems to play a role in protection of endothelial cells. The distinct difference in antioxidant protection against ethanol appears to depend on the capability of each cell to produce cytotoxic oxygen species in response to ethanol exposure.     

Alterations in Cyclic AMP Metabolism in Human Bronchial Asthma. III. LEUKOCYTE AND LYMPHOCYTE RESPONSES TO STEROIDS

On the basis of serial studies the responsiveness of leukocytes and lymphocytes from asthmatic donors to catecholamines was increased during high dose corticosteroid therapy. Similar changes were observed in the cells of normal control subjects given 200 mg of hydrocortisone intravenously. The increase in responsiveness did not appear to be due to changes in lymphocyte subpopulations although this may be a contributing factor. In an effort to elucidate the basis for the improved response, in vitro effects of glucocorticoids on lymphocyte cyclic AMP concentrations were investigated. Glucocorticoids (prednisolone succinate, hydrocortisone, hydrocortisone phosphate, and hydrocortisone succinate) stimulated cyclic AMP accumulation in asthma and normal control lymphocytes, increases occurring within the first 2 min of incubation. In the absence of theophylline, responses were regularly obtained at 10 muM hydrocortisone and usually at 1 muM hydrocortisone but not at submicromolar steroid concentrations. Theophylline potentiated the cyclic AMP response to glucocorticoids and also increased the percentage of positive responses in the 0.01-1.0 muM corticosteroid range. Combinations of 1 muM hydrocortisone and 1 muM epinephrine were sometimes additive or synergistic but in many instances higher glucocorticoid concentrations were needed to obtain augmentation of the catecholamine response. The in vitro glucocorticoid effects may not fully explain their potentiating action in vivo.     

In vitro detection of endothelial cell damage using 2-deoxy-D-3H-glucose: comparison with chromium 51, 3H-leucine, 3H-adenine, and lactate dehydrogenase

To develop a sensitive in vitro assay for detecting endothelial cell damage, we radiolabeled endothelial cell monolayers with tracer amounts of 2-deoxy-D-[1-3H]-glucose (3HDOG). We damaged identical cohorts of endothelial cells radiolabeled with 3HDOG or chromium 51 by exposing monolayers to toxic oxygen radicals generated by xanthine-xanthine oxidase or phorbol myristate acetate (PMA)-activated polymorphonuclear leukocytes (PMNs), a surface active agent (Triton X-100), and anti-HLA antibodies and complement. With each mechanism of injury, the 3HDOG assay detected significant (P less than 0.01) endothelial cell damage at lower concentrations of the injurious agent than the 51Cr assay. When endothelial monolayers were damaged by xanthine-xanthine oxidase or PMA-activated PMNs, efflux of 3HDOG was reduced (range 71% to 94% reduction) by superoxide dismutase and catalase, indicating that efflux of 3HDOG was mediated by toxic oxygen radicals. When monolayers were damaged with xanthine oxidase in the absence of glucose, a much lower concentration of xanthine oxidase was required to initiate efflux of 3HDOG as compared with xanthine oxidase injury in the presence of glucose. Additional studies compared the 3HDOG assay with 3H-adenine, 3H-leucine, and lactate dehydrogenase (LDH) release when endothelial cells were exposed to toxic oxygen radicals generated by PMA-activated PMNs and xanthine-xanthine oxidase. Again, the 3HDOG assay was more sensitive in detecting in vitro endothelial cell damage. We conclude that the 3HDOG assay is more sensitive than the 51Cr, 3H-adenine, 3H-leucine, or LDH release assays in detecting endothelial cell damage in vitro.     

Mechanisms of anti-inflammatory action of dexamethasone: blockade by hydrocortisone mesylate and actinomycin D of the inhibitory effect of dexamethasone on leukocyte infiltration in inflammatory sites

The present study was designed to clarify molecular mechanisms underlying inhibitory effect of dexamethasone on leukocyte infiltration in the inflammatory site. For the assay of leukocyte infiltration, two or four blebs were made s.c. on the back of rats by injecting with 2% carboxymethyl cellulose solution containing a chemoattractant, casein. Leukocyte accumulation in the bleb was inhibited considerably by local application of dexamethasone at a concentration of 0.6 X 10(-6) M. Hydrocortisone mesylate, which was reported in the study with hepatoma tissue culture cells to be a long-acting antagonist against glucocorticoid in binding to the corticoid receptor, blocked the above leukocyte inhibitory effect of dexamethasone when applied simultaneously with dexamethasone. The leukocyte infiltration was unaffected by the application of hydrocortisone mesylate alone. Treatment with androstenedione, which was reported to be inactive in the hepatoma tissue culture cells, did not interfere with the inhibitory effect of dexamethasone at all. Actinomycin D, when applied simultaneously with dexamethasone, significantly suppressed the leukocyte-inhibitory effect of dexamethasone. In contrast with those observations, the inhibitory effect of dexamethasone was not affected at all in cases that actinomycin D and hydrocortisone mesylate, respectively, were applied after the administration of dexamethasone. These results indicate essential roles of glucocorticoid receptor and gene expression for the manifestation of the inhibitory effect of dexamethasone on leukocyte infiltration in the inflammatory site.     

Injury to human endothelial cells in culture induced by low density lipoproteins.

Low density lipoproteins (LDL) have been shown to injure culture endothelial cells derived from the human umbilical cord. During a 48 h incubation period LDL significantly increased 51Cr release from prelabelled cells and induced marked cellular injury if the ratio between the LDL cholesterol and the infranatant proteins was kept above 0.1-0.12 mmol/g protein. Actually, an injurious effect of a fixed concentration of LDL could be completely prevented by increasing the concentration of infranatant proteins. High density lipoproteins within physiological concentration ranges had no effect when tested in the presence of infranatant proteins. The effects of LDL were not cell specific because normal as well as LDL receptor negative human skin fibroblasts were injured by LDL.     

Liquid-chromatographic measurement of endogenous and exogenous glucocorticoids in plasma.

The therapeutic response to and side effects of glucocorticoids will be better recognized and the recovery of the adrenals during the tapering of therapy with steroids better evaluated if endogenous and exogenous glucocorticoids are separately assessed. We describe a specific method for simultaneously measuring the concentrations of cortisone, cortisol, prednisone, and prednisolone in plasma by "high-pressure" liquid chromatography. The steroids, together with an internal standard, dexamethasone, are extracted from 1 mL of plasma with methylene chloride-ether, washed with acid and base, and separated isocratically on a normal-phase silica column with a mobile phase consisting of methylene chloride/tetrahydrofuran/methanol/glacial acetic acid (96.85/1/2.1/0.05 by vol) at a flow rate of 1.3 mL/min. The steroids are detected at 254 nm and quantitated by peak-height measurements; their retention times range from 6 to 20 min. The lower limits for routine detection of all four compounds is 10 microgram/L. The analytical recoveries are about 75%; the intra-day variability (CV) is 1 to 9%, and the inter-day variability 2 to 11%. Of 26 drugs and 20 steroids tested, only theophylline presents an interference problem.     

Selective growth of epithelial-like clear cells from adult rat liver by short-term exposure to glucocorticoids in primary culture

Isolated liver cells, which were prepared from adult rats by a trypsin-liver-perfusion technique, were treated with dexamethasone or hydrocortisone at a concentration of 7.7 X 10(-6) M for 8 days in primary culture. The treated cultures displayed homogeneous population consisting of epithelial-like clear cells, while the untreated cultures displayed mixed population consisting of epithelial-like clear cells and fibroblast-like cells. The epithelial-like clear cells, which proliferated in the cultures treated with glucocorticoids for 8 days in primary culture, did not show any morphological changes following cultivation in glucocorticoid-free medium. After continuous glucocorticoid-treatment for more than 1 month, the treated cultures showed relatively low cell densities at confluence. The surface area of individual epithelial-like clear cells in the cultures treated with glucocorticoids for long periods of time was evidently greater than that in the cultures treated for only 8 days. The epithelial-like clear cells had glucose 6-phosphatase and tyrosine aminotransferase activities even though the levels of these enzyme-activities were very low compared with those in rat liver homogenates.     

Effect of dexamethasone on IL-1 and IL-3-LA release by unstimulated human mononuclear cells

The effect of dexamethasone on the in vitro release of IL-1 and IL-3-LA by human unstimulated mononuclear cells was studied. The drug elicited a dose-dependent effect on the production of both interleukins. In addition, a time course effect of dexamethasone on IL-3-LA was demonstrated. It appeared after 5 min of incubation, reached a maximal level after 15 min, and remained at that level after 24 h. The drug also caused a dose-dependent inhibitory effect on the ability of human mononuclear cells to incorporate [3H] uridine. The inhibitory effect of dexamethasone on these interleukins may serve as a partial explanation of the susceptibility to infection in patients treated with corticosteroids.