Effect of very low LDL cholesterol levels on the steroid metabolome

Effect of very low LDL cholesterol levels on the steroid metabolome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of very low LDL cholesterol levels on the steroid metabolome Tereza Foglarová, Václav Hána, Drahomíra Springer, Marcela Kotasová, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7464305/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Recommended cholesterol levels are continuously being lowered according to clinical guidelines for cardiovascular prevention. Cholesterol is an essential substrate for steroidogenesis in the adrenal glands. Methods To evaluate steroid hormone production associated with intensive hypolipidemic therapy, we measured steroid levels at baseline and following a Synacthen (1–24 ACTH) stimulation test in 36 patients with persistently very low Low-density lipoprotein cholesterol (LDL-C) levels (< 1 mmol/L, < 38.7 mg/dL). Results All patients demonstrated a sufficient cortisol response to stimulation, which excluded partial or complete adrenal insufficiency. We observed correlations between various steroids and LDL-C, High-density lipoprotein cholesterol (HDL-C), and Triglyceride (TG) levels. Conclusions These results confirm the safety of intense hypolipidemic therapy even in patients with very low LDL cholesterol levels. Changes in TG, HDL-C and very low LDL-C cholesterol levels are associated with alterations in adrenal and gonadal steroidogenesis. cholesterol LDL HDL Synacthen test adrenal gland adrenocortical insufficiency Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Cardiovascular diseases are a major cause of morbidity and mortality. The recommended optimal levels for LDL cholesterol (LDL-C) are regularly reduced. The current 2024 ESC guidelines recommend LDL-C levels < 3 mmol/l for patients with low cardiovascular risk, < 2.6 mmol/l for those with moderate risk, < 1.8 mmol/l for those with high risk, and < 1 mmol/l for very high-risk patients(1,2). Several types of LDL-C–lowering drugs, as well as drugs targeting associated risk factors (triglycerides and lipoprotein (a)), have been developed to prevent atherosclerosis. Cholesterol is an essential precursor for the synthesis of steroid hormones(3). Adrenal glands obtain cholesterol in several ways. The two main sources of adrenal cholesterol in humans are LDL particles (via receptor-mediated endocytosis through endosomes) and de novo synthesis in the endoplasmic reticulum of adrenal cells(4). There is also accrual of HDL cholesterol (HDL-C) via scavenger receptor B type 1 (SR-B1), which is more potent in rodents than in humans, where its activity is considered minor compared to that of LDL-C. The retrieved or synthesized cholesterol is converted into pregnenolone, which is transformed into other steroid hormones(5). The question of whether low LDL-C levels after lipid-lowering therapy reduce the ability of the adrenal glands to produce steroid hormones in sufficient quantities has been discussed but not fully answered. Patients with abetalipoproteinemia, a congenital condition in which lipoprotein particles containing ApoB are absent due to a homozygous mutation in the microsomal triglyceride-transferring protein, have preserved steroid hormone function under basal conditions but a reduced adrenal response after the Synacthen test(6). Similar results, in which adrenal production was adequate under basal conditions but insufficient under increased stress, were observed in patients with heterozygous and homozygous hypobetalipoproteinemia in a study that examined adrenal function in patients with apolipoprotein B synthesis disorders(7). The impact of low LDL-C levels on adrenal and gonadal function has been tested not only in patients with rare lipoprotein disorders but also in patients whose low LDL-C levels are induced by lipid-lowering therapy. Patients receiving statin therapy had lower levels of SHBG and DHEAS(8). Testosterone and estradiol levels remained unchanged in this group of patients. Another study measured the effects of high-dose simvastatin on adrenal and gonadal steroidogenesis in men with hypercholesterolemia. After dietary modification and subsequent simvastatin therapy, serum testosterone levels decreased, and gonadotropin and SHBG levels remained unchanged(9). Studies focusing on the corticotroph axis demonstrated variable results. In the study by American authors entitled “Acute Statin Administration Reduces Levels of Steroid Hormone Precursors,” after a single dose of pitavastatin, there was a significant decrease in corticosterone, cortisol, and 11-deoxycortisol levels after 24 hours(10). Conversely, in another study measuring the effect of very low LDL-C levels on cortisol synthesis, no changes were observed in patients receiving intensive statin therapy(11). Similar studies have also been conducted to assess the safety of proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9is). A study evaluating the safety of evolocumab revealed reduced aldosterone but not cortisol or testosterone levels after PCSK9is therapy(12,13). Given the uncertain results of studies in patients with very low LDL-C levels, we aimed to test whether very low LDL-C levels can influence stress-induced cortisol levels as well as the levels of other steroid hormones under basal and stress-induced conditions. Methods This study included thirty-six male patientswith documentation of high-intensity hypolipidemic therapy and LDL-C levels of 0.5 ± 0.3 mmol/l (mean ± standard deviation) for at least one year. The baseline characteristics of the study participants are shown in Table 1. The exclusion criteria were as follows: corticotherapy, including local application, within the last year; known adrenal disease; the use of drugs affecting steroidogenesis; age less than 18 years; and female sex due to the influence of ovarian steroids. The local ethical committee approved the study. All participants signed an informed consent. This study adheres to the Declaration of Helsinki. Table 1: Baseline characteristics of the study group PCSK9i: Proprotein convertase subtilisin/kexin type 9 inhibitor Twenty-two patients were treated with secondary prevention, and 17 patients had confirmed mutations for familial hypercholesterolemia in either the homozygous (no null mutation) or heterozygous form. Three patients were euthyroid on replacement therapy, and 9 patients had type 2 diabetes mellitus. We performed adrenal stimulation in all patients with 10 μg of Synacthen (1-24 ACTH), with concurrent measurements of 17 steroid hormones at baseline (time 0) and at 30 and 60 minutes following stimulation with Synacthen. Additionally, we measured the baseline serum levels of ACTH, TSH, free T4, insulin, C-peptide, fasting glucose, and glycated hemoglobin. All steroid levels at baseline and poststimulation were determined using liquid chromatography‒mass spectrometry (LC‒MS), and basic steroid levels were also determined by routine immunoreaction-based methods(14). The results were statistically processed using a power transformation into a symmetric distribution with constant variance. We performed multivariate regression with dimensionality reduction (orthogonal projection to latent structure "OPLS"). In this model, we searched for inhomogeneities in the matrix of explanatory variables using the Hotelling T2 statistic and assessed the relevance of the explanatory variables using variable importance projection (VIP) statistics. The relevant explanatory variables were retained in the model. Finally, we evaluated the homogeneity of all the explained variables. Results All the subjects had a sufficient response to Synacthen stimulation. The average cortisol concentrations measured by LCMS at 0, 30, and 60 min were 401.9 nmol/l, 675.8 nmol/l and 634 nmol/l, respectively. The cortisol levels are shown in Figure 1. Other results are shown in Tables 2 and 3. Figure 1 Cortisol (LCMS) levels in the ACTH stimulation test Cortisol in nmol/L, shown as x: mean­: median, whiskers with minimal and maximal values, and boxes showing quartiles 1–3. Table 2 . Baseline LDL-C, HDL-C, TG, and steroid hormone levels and other results Steroid hormone levels measured by CLIA. Table 3 Steroid hormone levels measured by LCMS at baseline and after ACTH stimulation In addition to cortisol, we correlated other steroids and hormones with LDL-C, HDL-C and TG levels using the OPLS model. We observed a positive correlation between LDL-C and testosterone levels (measured by both CLIA and LCMS) and a negative correlation between LDL-C and cortisol levels at times 30 and 60, 11-deoxycorticosterone level at times 30 and 60, 21-deoxycortisol level at time 60, and 17-OH progesterone level at times 30 and 60 (and the ratio between 17-OH progesterone level at times 0-30 and 0-60). The OPLS model for LDL-C is shown in Figure 2. Figure 2 . Predictive components for LDL-C The OPLS model for LDL-C shows that steroid hormones that are statistically associated with LDL-C levels T– Testosterone (immunoanalysis); FLC_30 – Cortisol (LCMS), time 30; FLC_60 – Cortisol (LCMS), time 60; T_0 – Testosterone (LCMS), time 0; S_30 – 11-deoxycortisol (LCMS), time 30; S_60 – 11-deoxycortisol (LCMS), time 60; DOF_60 – 21-deoxycortisol (LCMS), time 60; P17_30 – 17-OHprogesterone (LCMS), time 30; P17_60 – 17-OHprogesterone (LCMS), time 60; P17_30_0 – 17-OHprogesterone (LCMS), in the ratio at times 0-30; P17_60_0 – 17-OHprogesterone (LCMS), in the ratio at times 0–60; LDL-C after a minimum of one year of extensive hypolipidemic therapy; **predictive component p<0.01 In relation to HDL-C levels and the selected steroid hormones, we observed positive correlations with androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17α-hydroxyprogesterone, cortisol at baseline (CLIA and LCMS), aldosterone and DHEA at time 0 and 60 min, DHEAS, 11-deoxycortisol, cortisone, 11-deoxycorticosterone, 17-OHprogesterone, and dihydrotestosterone at time 0. Conversely, a negative correlation was found between HDL-C levels and DM type 2 (C-peptide, insulin) and BMI. The ratios of cortisol, aldosterone, androstenedione, DHEA, 11-deoxycortisol, corticosterone, and cortisone increased at times 30–0 and 60–0. Additionally, a negative correlation was detected between HDL-C and the ratio of deoxycorticosterone to 17-OHprogesterone (at time 30–0). Figure 3 . Predictive components for HDL cholesterol The OPLS model for HDL -C shows the presence of DM, BMI, insulin, C-peptide and steroid hormone levels that are statistically associated with HDL-C levels; positive and negative correlations with HDL -C DM – Diabetes mellitus type 2 (in anamnesis); BMI – Body mass index (calculation); Insulin – Insulin (ECLIA); C-peptide (ECLIA); A – Androstenedione (CLIA); DHEA – Dehydroepiandrosterone (RIA); DHEAS – Dehydroepiandrosterone sulfate (CLIA); P17 – 17-OHprogesterone (CLIA); F_IA – Cortisol (CLIA); FLC_0 – Cortisol (LCMS), time 0; FLC_30_0 – Cortisol (LCMS), ratio of times 0-30; FLC_60_0 – Cortisol (LCMS), ratio of times 0-60; Aldo_0 – Aldosterone (LCMS), time 0; Aldo_60 – Aldosterone (LCMS), time 60; A_30_0 – Androstenedione (LCMS), ratio of times 0–30; A_60_0 – Androstenedione (LCMS), ratio of times 0–60; DHEA_0 – Dehydroepiandrosterone (LCMS), time 0; DHEA_60 – Dehydroepiandrosterone (LCMS), time 60; DHEA_30_0 – Dehydroepiandrosterone (LCMS), in the ratio of times 0–30; DHEA_60_0 – Dehydroepiandrosterone (LCMS), in the ratio of times 0–60; DHEAS_0 – Dehydroepiandrosterone sulfate (LCMS), time 0; S_0 – 11-deoxycortisol (LCMS), time 0; S_30_0 – 11-deoxycortisol (LCMS), ratio of times 0–30; S_60_0 – 11-deoxycortisol (LCMS), ratio of times 0–60; B_0 – Corticosterone (LCMS), time 0; B_30_0 – Corticosterone (LCMS), ratio of times 0–30; B_60_0 – Corticosterone (LCMS), ratio of times 0–60; E_0 – Cortisone (LCMS), time 0; E_30_0 – Cortisone (LCMS), ratio of times 0–30; E_60_0 – Cortisone (LCMS), ratio of times 0–60; DOC_0 – Deoxycorticosterone (LCMS), time 0; DOC_30_0 – Deoxycorticosterone (LCMS), ratio of times 0–30; P17_0 – 17OH-progesterone (LCMS), time 0; P17_30_0 – 17OH-progesterone (LCMS), ratio of times 0–30; DHT_0 – Dihydrotestosterone (LCMS), time 0; *predictive component p<0,05; ** predictive component p<0,01 The third model shows a correlation between related steroids and triacylglycerol (TG). A positive correlation was observed between TG and insulin levels, C-peptide levels and glycemia. A mild negative correlation was observed with aldosterone at time 30, testosterone, dihydrotestosterone and stimulated corticosterone at times 30 and 60. Figure 4 . Predictive components for TG The OPLS model for TG shows that steroid hormones, insulin, C-peptide and glycemia levels are statistically associated with TG levels and are positively and negatively correlated with TG Aldo_30 – aldosterone (LCMS), time 30; T_0 – testosterone (LCMS), time 0; B_30 – corticosterone (LCMS), time 30; B_60 – corticosterone (LCMS), time 60; DHT_0 – dihydrotestosterone (LCMS), time 0; TG – triglycerides after a minimum of one year of extensive hypolipidemic therapy; *predictive component p<0.05; **predictive component p<0.01 Discussion The physiological cortisol response in all patients confirmed the safety of hypolipidemic therapy. Additionally, other adrenal and gonadal steroids remained in physiological ranges. Since steroids in humans are produced mainly from LDL-C but also from HDL-C and de novo synthesis, we examined the correlations between LDL-C, HDL-C and TG levels with measured variables and steroids to determine whether steroidogenesis can be linked to LDL-C, HDL-C or TG levels(4). In the OPLS model, the LDL-C level was positively correlated with testosterone. Although many studies have not proven any association between cholesterol levels/statin use and total testosterone, data from patients with very low LDL-C levels are lacking. Compared with placebo, evolocumab therapy did not result in a decrease in testosterone(12). Our results showing a positive correlation between LDL-C and testosterone levels could be explained by more intense hypolipidemic therapy in patients with comorbidities and associated lower testosterone(9). Cortisol-stimulated levels were inversely correlated with LDL-C levels, so subjects with higher LDL-C levels had lower stimulated levels of cortisol but were still in the physiological range. Additionally, the levels of cortisol precursors, such as 11-deoxycortisol or 17-OHprogesterone, were inversely correlated with LDL-C levels. These findings suggest that adrenal steroidogenesis is not LDL-C dependent even at very low LDL-C levels. HDL-C levels were positively correlated with baseline levels of most steroids, such as cortisol, DHEA, DHEAS, aldosterone, 17-hydroxyprogesterone, and dihydrotestosterone. On the other hand, diabetes mellitus (incl. C-peptide, insulin), BMI and several stimulated steroid ratios (increments after ACTH stimulation) were inversely associated with HDL-C levels. The pathophysiology of this phenomenon is not fully understood, but insulin resistance with a secondary alteration in lipid metabolism is likely the main factor(15). We hypothesize that the presence of diabetes mellitus favors lower HDL-C levels, lower DHT levels, and lower basal cortisol and other steroid levels but higher cortisol and other steroid levels after ACTH stimulation than in nondiabetic subjects. Similarly, lower baseline cortisol levels were associated with lower HDL-C levels in a study by Bochem et al., which suggests a possible HDL substrate for basal steroidogenesis but not under stress (ACTH)-stimulated conditions(16). TG levels were positively related to C-peptide, insulin and glucose levels but negatively correlated with testosterone and DHT levels. This finding suggests a strong association between obesity and elevated TG levels, insulin resistance and a decrease in testosterone(17). Taken together, all three OPLS models indicate that higher TG levels are associated with a greater probability of diabetes mellitus and low testosterone levels. LDL-C and HDL-C levels are closely related to adrenal steroidogenesis, whereas baseline steroidogenesis levels are strongly correlated with HDL-C. The increase induced by stress (ACTH) is inversely related to both LDL-C and HDL-C levels, so it probably does not serve as a major substrate after stimulation. A few drawbacks of this study need to be mentioned. The measurement of baseline steroids before the initiation of lipid-lowering therapy would be beneficial. The study group was acquired consecutively over a long period and was not homogeneous in terms of hypolipidemic treatment, which could have altered the evaluation. Additionally, steroid levels before and after hypolipidemic treatment could shed more light on causality and not only associations between steroid hormones and LDL-C, HDL-C, and TG levels. Conclusion Long-term hypolipidemic therapy targeting LDL-C does not significantly affect cortisol secretion. Low LDL-C, HDL-C and TG levels are associated with steroid hormone changes that can be caused by altered adrenal and gonadal steroidogenesis. Abbreviations A Androstenedione Aldo Aldosterone B Corticosterone BMI Body mass index CLIA Chemiluminescence immunoassay DHEA Dehydroepiandrosterone DHEAS Dehydroepiandrosterone sulfate DHT Dihydrotestosterone DM Diabetes mellitus DOC Deoxycorticosterone DOF − 21 deoxycortisol E Cortisone ECLIA Electrochemiluminescence Immunoassay F_IA Cortisol (CLIA) FLC Cortisol (LCMS) HDL C–High–density lipoprotein cholesterol LCMS Liquid chromatography–mass spectrometry LDL C–Low–density lipoprotein cholesterol P Predictive component PCSK9i Proprotein convertase subtilisin/kexin type 9 inhibitor P17 17–OHprogesterone RIA Radioimmunoassay S 11–deoxycortisol T Testosterone TG Triglyceride VIP Variable importance projection statistics Declarations Ethical Approval and consent to participate: The study was approved by the regional ethics committee (Ethics Committee of the General University Hospital in Prague, č.j. 97/23). Clinical trial number: not applicable Consent to Publication: not applicable Data Availability statement: not applicable Conflict of interest: none Funding: This work was supported by a GIP-24-SL-03-203 internal grant and Cooperatio Program, research area “Endocrinology” 207037 and GJIH-1599-04-1-180 (supported by the Ministry of Health, Czech Republic - conceptual development of research organization 64165, General University Hospital in Prague, Czech Republic). Acknowledgment: not applicable Author contribution: T.F. and V.H. main manuscript and acquisition, analysis, M.H. and T.B.interpretation of data, D.S. and M.K. analysis, L.Z. design of the work. All authors reviewed the manuscript. Author information: not applicable References Visseren FLJ, Mach F, Smulders YM, Carballo D, Koskinas KC, Bäck M, et al. 2021 ESC Guidelines on cardiovascular disease prevention in clinical practice. Eur Heart J [Internet]. 2021 Sep 7;42(34):3227–337. 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Relationships between endogenous steroid hormone, sex hormone-binding globulin and lipoprotein levels in men: Contribution of visceral obesity, insulin levels and other metabolic variables. Atherosclerosis [Internet]. 1997 Sep 1 [cited 2025 Apr 1];133(2):235–44. Available from: https://www.atherosclerosis-journal.com/action/showFullText?pii=S0021915097001251 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 16 Oct, 2025 Reviewers agreed at journal 05 Oct, 2025 Reviewers invited by journal 03 Oct, 2025 Editor invited by journal 12 Sep, 2025 Editor assigned by journal 03 Sep, 2025 Submission checks completed at journal 02 Sep, 2025 First submitted to journal 02 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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2","display":"","copyAsset":false,"role":"figure","size":168176,"visible":true,"origin":"","legend":"\u003cp\u003ePredictivecomponents for LDL-C\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7464305/v1/12b84401000427d843ebdd6e.jpg"},{"id":93804721,"identity":"d4a521db-1bb6-413a-bf1f-4ec34338ab62","added_by":"auto","created_at":"2025-10-17 17:51:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":115308,"visible":true,"origin":"","legend":"\u003cp\u003ePredictivecomponents for HDL cholesterol\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7464305/v1/14c2129fa859d3a7a7e65e26.jpg"},{"id":93804726,"identity":"2e34bd5c-ef98-42da-8f2c-73098bd38f44","added_by":"auto","created_at":"2025-10-17 17:51:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":154744,"visible":true,"origin":"","legend":"\u003cp\u003ePredictivecomponents for TG\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7464305/v1/988cbcc4416f690a80b2d49d.jpg"},{"id":93806667,"identity":"3d1ea38e-0c92-47e8-83b2-e36cf5045895","added_by":"auto","created_at":"2025-10-17 18:23:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1644720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7464305/v1/3ed1baa7-6213-4575-a106-a14d50a4e0cb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of very low LDL cholesterol levels on the steroid metabolome","fulltext":[{"header":"Background","content":"\u003cp\u003eCardiovascular diseases are a major cause of morbidity and mortality. The recommended optimal levels for LDL cholesterol (LDL-C) are regularly reduced. The current 2024 ESC guidelines recommend LDL-C levels\u0026thinsp;\u0026lt;\u0026thinsp;3 mmol/l for patients with low cardiovascular risk, \u0026lt; 2.6 mmol/l for those with moderate risk, \u0026lt; 1.8 mmol/l for those with high risk, and \u0026lt;\u0026thinsp;1 mmol/l for very high-risk patients(1,2). Several types of LDL-C\u0026ndash;lowering drugs, as well as drugs targeting associated risk factors (triglycerides and lipoprotein (a)), have been developed to prevent atherosclerosis.\u003c/p\u003e\u003cp\u003eCholesterol is an essential precursor for the synthesis of steroid hormones(3). Adrenal glands obtain cholesterol in several ways. The two main sources of adrenal cholesterol in humans are LDL particles (via receptor-mediated endocytosis through endosomes) and de novo synthesis in the endoplasmic reticulum of adrenal cells(4). There is also accrual of HDL cholesterol (HDL-C) via scavenger receptor B type 1 (SR-B1), which is more potent in rodents than in humans, where its activity is considered minor compared to that of LDL-C.\u003c/p\u003e\u003cp\u003eThe retrieved or synthesized cholesterol is converted into pregnenolone, which is transformed into other steroid hormones(5). The question of whether low LDL-C levels after lipid-lowering therapy reduce the ability of the adrenal glands to produce steroid hormones in sufficient quantities has been discussed but not fully answered.\u003c/p\u003e\u003cp\u003ePatients with abetalipoproteinemia, a congenital condition in which lipoprotein particles containing ApoB are absent due to a homozygous mutation in the microsomal triglyceride-transferring protein, have preserved steroid hormone function under basal conditions but a reduced adrenal response after the Synacthen test(6). Similar results, in which adrenal production was adequate under basal conditions but insufficient under increased stress, were observed in patients with heterozygous and homozygous hypobetalipoproteinemia in a study that examined adrenal function in patients with apolipoprotein B synthesis disorders(7).\u003c/p\u003e\u003cp\u003eThe impact of low LDL-C levels on adrenal and gonadal function has been tested not only in patients with rare lipoprotein disorders but also in patients whose low LDL-C levels are induced by lipid-lowering therapy. Patients receiving statin therapy had lower levels of SHBG and DHEAS(8). Testosterone and estradiol levels remained unchanged in this group of patients. Another study measured the effects of high-dose simvastatin on adrenal and gonadal steroidogenesis in men with hypercholesterolemia. After dietary modification and subsequent simvastatin therapy, serum testosterone levels decreased, and gonadotropin and SHBG levels remained unchanged(9). Studies focusing on the corticotroph axis demonstrated variable results. In the study by American authors entitled \u0026ldquo;Acute Statin Administration Reduces Levels of Steroid Hormone Precursors,\u0026rdquo; after a single dose of pitavastatin, there was a significant decrease in corticosterone, cortisol, and 11-deoxycortisol levels after 24 hours(10). Conversely, in another study measuring the effect of very low LDL-C levels on cortisol synthesis, no changes were observed in patients receiving intensive statin therapy(11).\u003c/p\u003e\u003cp\u003eSimilar studies have also been conducted to assess the safety of proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9is). A study evaluating the safety of evolocumab revealed reduced aldosterone but not cortisol or testosterone levels after PCSK9is therapy(12,13).\u003c/p\u003e\u003cp\u003eGiven the uncertain results of studies in patients with very low LDL-C levels, we aimed to test whether very low LDL-C levels can influence stress-induced cortisol levels as well as the levels of other steroid hormones under basal and stress-induced conditions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis study included thirty-six male patientswith documentation of high-intensity hypolipidemic therapy and LDL-C levels of 0.5\u0026nbsp;±\u0026nbsp;0.3 mmol/l (mean ± standard deviation) for at least one year. \u0026nbsp; The baseline characteristics of the study participants are shown in Table 1. The exclusion criteria were as follows: corticotherapy, including local application, within the last year; known adrenal disease; the use of drugs affecting steroidogenesis; age less than 18 years; and female sex due to the influence of ovarian steroids. The local ethical committee approved the study. All participants signed an informed consent. This study adheres to the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Baseline characteristics of the study group\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"425\" height=\"374\" 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alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003ePCSK9i: Proprotein convertase subtilisin/kexin type 9 inhibitor\u003c/p\u003e\n\u003cp\u003eTwenty-two patients were treated with secondary prevention, and 17 patients had confirmed mutations for familial hypercholesterolemia in either the homozygous (no null mutation) or heterozygous form. Three patients were euthyroid on replacement therapy, and 9 patients had type 2 diabetes mellitus.\u003c/p\u003e\n\u003cp\u003eWe performed adrenal stimulation in all patients with 10 μg of Synacthen (1-24 ACTH), with concurrent measurements of 17 steroid hormones at baseline (time 0) and at 30 and 60 minutes following stimulation with Synacthen. Additionally, we measured the baseline serum levels of ACTH, TSH, free T4, insulin, C-peptide, fasting glucose, and glycated hemoglobin. All steroid levels at baseline and poststimulation were determined using liquid chromatography‒mass spectrometry (LC‒MS), and basic steroid levels were also determined by routine immunoreaction-based methods(14).\u003c/p\u003e\n\u003cp\u003eThe results were statistically processed using a power transformation into a symmetric distribution with constant variance. We performed multivariate regression with dimensionality reduction (orthogonal projection to latent structure \"OPLS\"). In this model, we searched for inhomogeneities in the matrix of explanatory variables using the Hotelling T2 statistic and assessed the relevance of the explanatory variables using variable importance projection (VIP) statistics. The relevant explanatory variables were retained in the model. Finally, we evaluated the homogeneity of all the explained variables.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAll the subjects had a sufficient response to Synacthen stimulation. The average cortisol concentrations measured by LCMS at 0, 30, and 60 min were 401.9 nmol/l, 675.8 nmol/l and 634 nmol/l, respectively. The cortisol levels are shown in Figure 1. Other results are shown in Tables 2 and 3.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFigure 1\u003c/strong\u003e Cortisol (LCMS) levels in the ACTH stimulation test\u003c/p\u003e\n\u003cp\u003eCortisol in nmol/L, shown as x: mean\u0026shy;: median, whiskers with minimal and maximal values, and boxes showing quartiles 1\u0026ndash;3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Baseline LDL-C, HDL-C, TG, and steroid hormone levels and other results\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"605\" height=\"251\" 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alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eSteroid hormone levels measured by CLIA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Steroid hormone levels measured by LCMS at baseline and after ACTH stimulation\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"549\" height=\"337\" 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alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to cortisol, we correlated other steroids and hormones with LDL-C, HDL-C and TG levels using the OPLS model.\u003c/p\u003e\n\u003cp\u003eWe observed a positive correlation between LDL-C and testosterone levels (measured by both CLIA and LCMS) and a negative correlation between LDL-C and cortisol levels at times 30 and 60, 11-deoxycorticosterone level at times 30 and 60, 21-deoxycortisol level at time 60, and 17-OH progesterone level at times 30 and 60 (and the ratio between 17-OH progesterone level at times 0-30 and 0-60). The OPLS model for LDL-C is shown in Figure 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2\u003c/strong\u003e. Predictive components for LDL-C\u003c/p\u003e\n\u003cp\u003eThe \u003cstrong\u003eOPLS model for LDL-C shows\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethat\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esteroid hormones that are statistically associated with LDL-C levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eT\u0026ndash; Testosterone (immunoanalysis); FLC_30 \u0026ndash; Cortisol (LCMS), time 30; FLC_60 \u0026ndash; Cortisol (LCMS), time 60; T_0 \u0026ndash; Testosterone (LCMS), time 0; S_30 \u0026ndash; 11-deoxycortisol (LCMS), time 30; S_60 \u0026ndash; 11-deoxycortisol (LCMS), time 60; DOF_60 \u0026ndash; 21-deoxycortisol (LCMS), time 60; P17_30 \u0026ndash; 17-OHprogesterone (LCMS), time 30; P17_60 \u0026ndash; 17-OHprogesterone (LCMS), time 60; P17_30_0 \u0026ndash; 17-OHprogesterone (LCMS), in the ratio at times 0-30; P17_60_0 \u0026ndash; 17-OHprogesterone (LCMS), in the ratio at times 0\u0026ndash;60; LDL-C after\u0026nbsp;\u003c/em\u003e\u003cem\u003ea minimum of\u003c/em\u003e\u003cem\u003e\u0026nbsp;one year of extensive hypolipidemic therapy; **predictive component p\u0026lt;0.01\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn relation to HDL-C levels and the selected steroid hormones, we observed positive correlations with androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17\u0026alpha;-hydroxyprogesterone, cortisol at baseline (CLIA and LCMS), aldosterone and DHEA at time 0 and 60 min, DHEAS, 11-deoxycortisol, cortisone, 11-deoxycorticosterone, 17-OHprogesterone, and dihydrotestosterone at time 0.\u003c/p\u003e\n\u003cp\u003eConversely, a negative correlation was found between HDL-C levels and DM type 2 (C-peptide, insulin) and BMI. The ratios of cortisol, aldosterone, androstenedione, DHEA, 11-deoxycortisol, corticosterone, and cortisone increased at times 30\u0026ndash;0 and 60\u0026ndash;0. Additionally, a negative correlation was detected between HDL-C and the ratio of deoxycorticosterone to 17-OHprogesterone (at time 30\u0026ndash;0).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3\u003c/strong\u003e. Predictive components for HDL cholesterol\u003c/p\u003e\n\u003cp\u003eThe \u003cstrong\u003eOPLS model for HDL\u003c/strong\u003e\u003cstrong\u003e-C\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;shows\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epresence of DM, BMI, insulin, C-peptide and steroid hormone levels that are statistically associated with HDL-C levels; positive and negative\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecorrelations\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;with HDL\u003c/strong\u003e\u003cstrong\u003e-C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDM \u0026ndash; Diabetes mellitus type 2 (in anamnesis); BMI \u0026ndash; Body mass index (calculation); Insulin \u0026ndash; Insulin (ECLIA); C-peptide (ECLIA); A \u0026ndash; Androstenedione (CLIA); DHEA \u0026ndash; Dehydroepiandrosterone (RIA); DHEAS \u0026ndash; Dehydroepiandrosterone sulfate (CLIA); P17 \u0026ndash; 17-OHprogesterone (CLIA); F_IA \u0026ndash; Cortisol (CLIA); FLC_0 \u0026ndash; Cortisol (LCMS), time 0; FLC_30_0 \u0026ndash; Cortisol (LCMS), ratio of times 0-30; FLC_60_0 \u0026ndash; Cortisol (LCMS), ratio of times 0-60; Aldo_0 \u0026ndash; Aldosterone (LCMS), time 0; Aldo_60 \u0026ndash; Aldosterone (LCMS), time 60; A_30_0 \u0026ndash; Androstenedione (LCMS), ratio of times 0\u0026ndash;30; A_60_0 \u0026ndash; Androstenedione (LCMS), ratio of times 0\u0026ndash;60; DHEA_0 \u0026ndash; Dehydroepiandrosterone (LCMS), time 0; DHEA_60 \u0026ndash; Dehydroepiandrosterone (LCMS), time 60; DHEA_30_0 \u0026ndash; Dehydroepiandrosterone (LCMS), in the ratio of times 0\u0026ndash;30; DHEA_60_0 \u0026ndash; Dehydroepiandrosterone (LCMS), in the ratio of times 0\u0026ndash;60; DHEAS_0 \u0026ndash; Dehydroepiandrosterone sulfate (LCMS), time 0; S_0 \u0026ndash; 11-deoxycortisol (LCMS), time 0; S_30_0 \u0026ndash; 11-deoxycortisol (LCMS), ratio of times 0\u0026ndash;30; S_60_0 \u0026ndash; 11-deoxycortisol (LCMS), ratio of times 0\u0026ndash;60; B_0 \u0026ndash; Corticosterone (LCMS), time 0; B_30_0 \u0026ndash; Corticosterone (LCMS), ratio of times 0\u0026ndash;30; B_60_0 \u0026ndash; Corticosterone (LCMS), ratio of times 0\u0026ndash;60; E_0 \u0026ndash; Cortisone (LCMS), time 0; E_30_0 \u0026ndash; Cortisone (LCMS), ratio of times 0\u0026ndash;30; E_60_0 \u0026ndash; Cortisone (LCMS), ratio of times 0\u0026ndash;60; DOC_0 \u0026ndash; Deoxycorticosterone (LCMS), time 0; DOC_30_0 \u0026ndash; Deoxycorticosterone (LCMS), ratio of times 0\u0026ndash;30; P17_0 \u0026ndash; 17OH-progesterone (LCMS), time 0; P17_30_0 \u0026ndash; 17OH-progesterone (LCMS), ratio of times 0\u0026ndash;30; DHT_0 \u0026ndash; Dihydrotestosterone (LCMS), time 0; *predictive component p\u0026lt;0,05; ** predictive component p\u0026lt;0,01\u003c/p\u003e\n\u003cp\u003eThe third model shows a correlation between related steroids and triacylglycerol (TG).\u003c/p\u003e\n\u003cp\u003eA positive correlation was observed between TG and insulin levels, C-peptide levels and glycemia. A mild negative correlation was observed with aldosterone at time 30, testosterone, dihydrotestosterone and stimulated corticosterone at times 30 and 60.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4\u003c/strong\u003e. Predictive components for TG\u003c/p\u003e\n\u003cp\u003eThe \u003cstrong\u003eOPLS model for TG shows\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethat\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esteroid hormones, insulin, C-peptide and glycemia levels are statistically associated with TG levels\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and are positively and negatively correlated\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;with TG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAldo_30 \u0026ndash;\u0026nbsp;\u003c/em\u003e\u003cem\u003ealdosterone\u003c/em\u003e\u003cem\u003e\u0026nbsp;(LCMS), time 30; T_0 \u0026ndash;\u0026nbsp;\u003c/em\u003e\u003cem\u003etestosterone\u003c/em\u003e\u003cem\u003e\u0026nbsp;(LCMS), time 0; B_30 \u0026ndash;\u0026nbsp;\u003c/em\u003e\u003cem\u003ecorticosterone\u003c/em\u003e\u003cem\u003e\u0026nbsp;(LCMS), time 30; B_60 \u0026ndash;\u0026nbsp;\u003c/em\u003e\u003cem\u003ecorticosterone\u003c/em\u003e\u003cem\u003e\u0026nbsp;(LCMS), time 60; DHT_0 \u0026ndash;\u0026nbsp;\u003c/em\u003e\u003cem\u003edihydrotestosterone\u003c/em\u003e\u003cem\u003e\u0026nbsp;(LCMS), time 0; TG \u0026ndash;\u0026nbsp;\u003c/em\u003e\u003cem\u003etriglycerides\u003c/em\u003e\u003cem\u003e\u0026nbsp;after\u0026nbsp;\u003c/em\u003e\u003cem\u003ea minimum of\u003c/em\u003e\u003cem\u003e\u0026nbsp;one year of extensive hypolipidemic therapy; *predictive component p\u0026lt;0.05; **predictive component p\u0026lt;0.01\u003c/em\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe physiological cortisol response in all patients confirmed the safety of hypolipidemic therapy. Additionally, other adrenal and gonadal steroids remained in physiological ranges.\u003c/p\u003e\u003cp\u003eSince steroids in humans are produced mainly from LDL-C but also from HDL-C and de novo synthesis, we examined the correlations between LDL-C, HDL-C and TG levels with measured variables and steroids to determine whether steroidogenesis can be linked to LDL-C, HDL-C or TG levels(4).\u003c/p\u003e\u003cp\u003eIn the OPLS model, the LDL-C level was positively correlated with testosterone. Although many studies have not proven any association between cholesterol levels/statin use and total testosterone, data from patients with very low LDL-C levels are lacking. Compared with placebo, evolocumab therapy did not result in a decrease in testosterone(12). Our results showing a positive correlation between LDL-C and testosterone levels could be explained by more intense hypolipidemic therapy in patients with comorbidities and associated lower testosterone(9).\u003c/p\u003e\u003cp\u003eCortisol-stimulated levels were inversely correlated with LDL-C levels, so subjects with higher LDL-C levels had lower stimulated levels of cortisol but were still in the physiological range. Additionally, the levels of cortisol precursors, such as 11-deoxycortisol or 17-OHprogesterone, were inversely correlated with LDL-C levels. These findings suggest that adrenal steroidogenesis is not LDL-C dependent even at very low LDL-C levels.\u003c/p\u003e\u003cp\u003eHDL-C levels were positively correlated with baseline levels of most steroids, such as cortisol, DHEA, DHEAS, aldosterone, 17-hydroxyprogesterone, and dihydrotestosterone. On the other hand, diabetes mellitus (incl. C-peptide, insulin), BMI and several stimulated steroid ratios (increments after ACTH stimulation) were inversely associated with HDL-C levels. The pathophysiology of this phenomenon is not fully understood, but insulin resistance with a secondary alteration in lipid metabolism is likely the main factor(15). We hypothesize that the presence of diabetes mellitus favors lower HDL-C levels, lower DHT levels, and lower basal cortisol and other steroid levels but higher cortisol and other steroid levels after ACTH stimulation than in nondiabetic subjects. Similarly, lower baseline cortisol levels were associated with lower HDL-C levels in a study by Bochem et al., which suggests a possible HDL substrate for basal steroidogenesis but not under stress (ACTH)-stimulated conditions(16).\u003c/p\u003e\u003cp\u003eTG levels were positively related to C-peptide, insulin and glucose levels but negatively correlated with testosterone and DHT levels. This finding suggests a strong association between obesity and elevated TG levels, insulin resistance and a decrease in testosterone(17).\u003c/p\u003e\u003cp\u003eTaken together, all three OPLS models indicate that higher TG levels are associated with a greater probability of diabetes mellitus and low testosterone levels. LDL-C and HDL-C levels are closely related to adrenal steroidogenesis, whereas baseline steroidogenesis levels are strongly correlated with HDL-C. The increase induced by stress (ACTH) is inversely related to both LDL-C and HDL-C levels, so it probably does not serve as a major substrate after stimulation.\u003c/p\u003e\u003cp\u003eA few drawbacks of this study need to be mentioned. The measurement of baseline steroids before the initiation of lipid-lowering therapy would be beneficial. The study group was acquired consecutively over a long period and was not homogeneous in terms of hypolipidemic treatment, which could have altered the evaluation. Additionally, steroid levels before and after hypolipidemic treatment could shed more light on causality and not only associations between steroid hormones and LDL-C, HDL-C, and TG levels.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLong-term hypolipidemic therapy targeting LDL-C does not significantly affect cortisol secretion. Low LDL-C, HDL-C and TG levels are associated with steroid hormone changes that can be caused by altered adrenal and gonadal steroidogenesis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAndrostenedione\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAldo\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAldosterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCorticosterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBody mass index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCLIA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChemiluminescence immunoassay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDHEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDehydroepiandrosterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDHEAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDehydroepiandrosterone sulfate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDHT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDihydrotestosterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDiabetes mellitus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDOC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDeoxycorticosterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDOF \u0026minus;\u0026thinsp;21\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edeoxycortisol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCortisone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECLIA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eElectrochemiluminescence Immunoassay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eF_IA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCortisol (CLIA)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCortisol (LCMS)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHDL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eC\u0026ndash;High\u0026ndash;density lipoprotein cholesterol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLCMS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLiquid chromatography\u0026ndash;mass spectrometry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLDL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eC\u0026ndash;Low\u0026ndash;density lipoprotein cholesterol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePredictive component\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCSK9i\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProprotein convertase subtilisin/kexin type 9 inhibitor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP17\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e17\u0026ndash;OHprogesterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRIA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRadioimmunoassay\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e11\u0026ndash;deoxycortisol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTestosterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTriglyceride\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eVIP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eVariable importance projection statistics\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThe study was approved by the regional ethics committee (Ethics Committee of the General University Hospital in Prague, č.j. 97/23).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publication:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability statement:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003enone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp; This work was supported by a GIP-24-SL-03-203 internal grant and Cooperatio Program, research area “Endocrinology” 207037 and\u0026nbsp;GJIH-1599-04-1-180 (supported\u0026nbsp;by\u0026nbsp;the\u0026nbsp;Ministry of Health, Czech Republic - conceptual development of research organization 64165, General University Hospital in Prague, Czech Republic).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u003c/strong\u003e T.F. and V.H. main manuscript and acquisition, analysis, M.H. and T.B.interpretation of data, D.S. and M.K. analysis, L.Z. design of the work. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information:\u003c/strong\u003e not applicable\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVisseren FLJ, Mach F, Smulders YM, Carballo D, Koskinas KC, B\u0026auml;ck M, et al. 2021 ESC Guidelines on cardiovascular disease prevention in clinical practice. Eur Heart J [Internet]. 2021 Sep 7;42(34):3227\u0026ndash;337. Available from: https://academic.oup.com/eurheartj/article/42/34/3227/6358713\u003c/li\u003e\n\u003cli\u003eGrundy SM, Feingold KR. Guidelines for the Management of High Blood Cholesterol. Endotext [Internet]. 2022 May 28 [cited 2025 Jan 5]; Available from: https://www.ncbi.nlm.nih.gov/sites/books/NBK305897/\u003c/li\u003e\n\u003cli\u003eHu J, Zhang Z, Shen WJ, Azhar S. Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones. Nutr Metab (Lond) [Internet]. 2010 [cited 2025 Jan 5];7:47. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC2890697/\u003c/li\u003e\n\u003cli\u003eMiller WL, Auchus RJ. The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders. Endocr Rev [Internet]. 2010 Feb [cited 2025 Jan 5];32(1):81. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC3365799/\u003c/li\u003e\n\u003cli\u003ePoojari P, Paramanya A, Singh D, Ali A. Polycystic ovarian syndrome: Causes and therapies by herbal medicine. Herbal Medicines: A Boon for Healthy Human Life. 2022 Jan 1;435\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eIllingworth DR, Orwoll ES, Connor WE. Impaired cortisol secretion in abetalipoproteinemia. J Clin Endocrinol Metab [Internet]. 1980 May;50(5):977\u0026ndash;9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/6246140\u003c/li\u003e\n\u003cli\u003eILLINGWORTH DR, KENNY TA, ORWOLL ES. Adrenal Function in Heterozygous and Homozygous Hypobetalipoproteinemia*. J Clin Endocrinol Metab [Internet]. 1982 Jan;54(1):27\u0026ndash;33. Available from: https://academic.oup.com/jcem/article-lookup/doi/10.1210/jcem-54-1-27\u003c/li\u003e\n\u003cli\u003eOluleye OW, Kronmal RA, Folsom AR, Vaidya DM, Ouyang P, Duprez DA, et al. Association between Statin Use and Sex Hormone in the Multi-Ethnic Study of Atherosclerosis Cohort. Journal of Clinical Endocrinology and Metabolism. 2019;104(10):4600\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eDobs AS, Schrott H, Davidson MH, Bays H, Stein EA, Kush D, et al. Effects of high-dose simvastatin on adrenal and gonadal steroidogenesis in men with hypercholesterolemia. Metabolism [Internet]. 2000 Sep;49(9):1234\u0026ndash;8. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0026049500024562\u003c/li\u003e\n\u003cli\u003eLondon E, Tatsi C, Soldin SJ, Wassif CA, Backlund P, Ng D, et al. Acute Statin Administration Reduces Levels of Steroid Hormone Precursors. 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Available from: http://www.ncbi.nlm.nih.gov/pubmed/23511897\u003c/li\u003e\n\u003cli\u003eTchernof A, Labrie F, B\u0026eacute;langer A, Prud\u0026rsquo;Homme D, Bouchard C, Tremblay A, et al. Relationships between endogenous steroid hormone, sex hormone-binding globulin and lipoprotein levels in men: Contribution of visceral obesity, insulin levels and other metabolic variables. Atherosclerosis [Internet]. 1997 Sep 1 [cited 2025 Apr 1];133(2):235\u0026ndash;44. Available from: https://www.atherosclerosis-journal.com/action/showFullText?pii=S0021915097001251\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-endocrine-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bend","sideBox":"Learn more about [BMC Endocrine Disorders](http://bmcendocrdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bend/default.aspx","title":"BMC Endocrine Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cholesterol, LDL, HDL, Synacthen test, adrenal gland, adrenocortical insufficiency","lastPublishedDoi":"10.21203/rs.3.rs-7464305/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7464305/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e Recommended cholesterol levels are continuously being lowered according to clinical guidelines for cardiovascular prevention. Cholesterol is an essential substrate for steroidogenesis in the adrenal glands.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTo evaluate steroid hormone production associated with intensive hypolipidemic therapy, we measured steroid levels at baseline and following a Synacthen (1\u0026ndash;24 ACTH) stimulation test in 36 patients with persistently very low Low-density lipoprotein cholesterol (LDL-C) levels (\u0026lt;\u0026thinsp;1 mmol/L, \u0026lt; 38.7 mg/dL).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAll patients demonstrated a sufficient cortisol response to stimulation, which excluded partial or complete adrenal insufficiency. We observed correlations between various steroids and LDL-C, High-density lipoprotein cholesterol (HDL-C), and Triglyceride (TG) levels.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThese results confirm the safety of intense hypolipidemic therapy even in patients with very low LDL cholesterol levels. Changes in TG, HDL-C and very low LDL-C cholesterol levels are associated with alterations in adrenal and gonadal steroidogenesis.\u003c/p\u003e","manuscriptTitle":"Effect of very low LDL cholesterol levels on the steroid metabolome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 17:51:04","doi":"10.21203/rs.3.rs-7464305/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-10-16T13:08:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54972758634604752054868439395410791457","date":"2025-10-05T10:08:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-03T07:36:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-12T11:06:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-03T05:50:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T09:32:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Endocrine Disorders","date":"2025-09-02T09:28:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-endocrine-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bend","sideBox":"Learn more about [BMC Endocrine Disorders](http://bmcendocrdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bend/default.aspx","title":"BMC Endocrine Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"42dfc1aa-1f7d-4411-8497-9bce84b6e83e","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-17T17:51:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 17:51:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7464305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7464305","identity":"rs-7464305","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}