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Disease-specific sterols accumulate in the blood of patients with several rare lipid disorders. Biochemical measurement of these sterols is important for correct diagnosis and sometimes monitoring of treatment. Existing methods to measure sterols in blood, particularly plant sterols, are often laborious and time consuming. Partly as a result, clinical access to sterol measurements is limited in many parts of the world. A simple and rapid method to extract free sterols from human serum and quantitate their concentration using isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization was developed. The method was designed to be compatible with routine workflows (e.g., 96-well format) in a clinical lab and extensively validated. Serum from at least 125 controls were analyzed and used to estimate the upper reference limits for sitosterol, campesterol, stigmasterol, desmosterol, 7-dehydrocholesterol (7DHC), lathosterol, and cholestanol. Serum from patients with the rare lipid disorders sitosterolemia (n = 7), Smith-Lemli-Opitz syndrome (SLOS; n = 1), and cerebrotendinous xanthomatosis (CTX; n = 1) were analyzed. All seven sitosterolemia patients had greatly elevated levels of free plant sterols (sitosterol, campesterol, and stigmasterol) compared to the controls. The SLOS serum contained massively increased concentrations of 7DHC. CTX serum contained greatly increased concentrations of cholestanol, as well as 7DHC and lathosterol. Spiking experiments indicated that the method is likely also useful for the diagnosis of desmosterolosis and lathosterolosis. The reported method is a relatively simple and fast LC-MS/MS method capable of quantitating diagnostically important sterols and differentiated patients with three rare lipid disorders from controls., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: P. M. T.: Honoraria from Sanofi for lectures. E. E. A.: Honoraria from Amarin, Novartis and Sanofi for lectures. K. R.: Honoraria from Amgen, Amarin, NovoNordic, Novartis, Sanofi for lectures. A. B. W., S. R. D., and P. M. T.: Financial support from The South-Eastern Norway Regional Health Authority to the Hormone Laboratory (∼10,000€). The not-for-profit foundation “Stiftelsen Nils Normans forskningsfond til Hormonlaboratoriets fremme” has supported various research and development work at the Hormone Laboratory, including the development of submitted method. E. A. A., L. L. D., and K. R.: Financial support from The South-Eastern Norway Regional Health Authority to the Lipid Clinic (∼40,000€). Y. T. B., A. L. S., and U. D. declare no competing interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cerebrotendinous xanthomatosis (CTX) is caused by autosomal recessive loss-of-function mutations in CYP27A1, a gene encoding cytochrome p450 oxidase essential for bile acid synthesis, resulting in altered bile acid and lipid metabolism. Here, we aimed to identify metabolic aberrations that drive ongoing neurodegeneration in some patients with CTX despite chenodeoxycholic acid (CDCA) supplementation, the standard treatment in CTX. Using chromatographic separation techniques coupled to mass spectrometry, we analyzed 26 sterol metabolites in serum and cerebrospinal fluid (CSF) of patients with CTX and in one CTX brain. Comparing samples of drug naive patients to patients treated with CDCA and healthy controls, we identified 7α,12α-dihydroxycholest-4-en-3-one as the most prominently elevated metabolite in serum and CSF of drug naive patients. CDCA treatment substantially reduced or even normalized levels of all metabolites increased in untreated patients with CTX. Independent of CDCA treatment, metabolites of the 27-hydroxylation pathway were nearly absent in all patients with CTX. 27-hydroxylated metabolites accounted for ∼45% of total free sterol content in CSF of healthy controls but <2% in patients with CTX. Metabolic changes in brain tissue corresponded well with findings in CSF. Interestingly, 7α,12α-dihydroxycholest-4-en-3-one and 5α-cholestanol did not exert toxicity in neuronal cell culture. In conclusion, we propose that increased 7α,12α-dihydroxycholest-4-en-3-one and lack of 27-hydroxycholesterol may be highly sensitive metabolic biomarkers of CTX. As CDCA cannot reliably prevent disease progression despite reduction of most accumulated metabolites, supplementation of 27-hydroxylated bile acid intermediates or replacement of CYP27A1 might be required to counter neurodegeneration in patients with progressive disease despite CDCA treatment., Competing Interests: Conflicts of interest E. Y. is shareholder in CholesteniX Ltd.; Y. W. is listed as inventor on the patent Kit and method for quantitative detection of steroids US9851368B2 and is shareholder in CholesteniX Ltd.; P. B. D. is consultant at Retrophin and received an institutional grant from Retrophin; A. E. D. is consultant for Leadiant Biosciences and Retrophin, institutional grant recipient from Retrophin. The OHSU Foundation and Chemical Physiology and Biology Department have received gifts from Retrophin. These gifts, which have not been made specifically in connection with this research, have been reviewed by the OHSU integrity office; W. J. G. is listed as inventor on the patent Kit and method for quantitative detection of steroids US9851368B2 and is shareholder in CholesteniX Ltd. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cerebrotendinous xanthomatosis (CTX) and Lathosterolosis represent two treatable inherited disorders of cholesterol metabolism that are characterized by the accumulation of cholestanol and lathosterol, respectively. The age of the patients suspected of having these disorders is highly variable due to the very different phenotypes. The early diagnosis of these disorders is important because specific therapeutic treatment could prevent the disease progression. The biochemical diagnosis of these defects is generally performed analyzing the sterol profile. Since age-related levels of these sterols are lacking, this study aims to determine a preliminary comparison of plasma levels of cholestanol and lathosterol among Italian unaffected newborns, children and healthy adults., Methods: The sterols were extracted from 130 plasma samples (24 newborns, 33 children and 73 adults) by a liquid-liquid separation method and quantified by gas chromatography coupled with a flame ionization detector., Results: Cholesterol, cholestanol and lathosterol levels together with the cholestanol/cholesterol and lathosterol/cholesterol ratios are statistically different among the three groups. Cholesterol levels progressively increased from newborns to children and to adults, whereas cholestanol/cholesterol and cholestanol/lathosterol ratios progressively decreased from newborns to children and to adults. Lathosterol levels were higher in adults than in both newborns and children. In the total population a positive correlation was observed between cholesterol levels and both cholestanol (correlation coefficient = 0.290, p = 0.001) and lathosterol levels (correlation coefficient = 0.353, p <  0.0001)., Conclusions: Although this study can only be considered an explorative experience due to the low number of analyzed samples, we revealed several differences of plasma cholestanol and lathosterol levels and their ratios to cholesterol levels among newborns, children and adults. These evidences indicate the need of age-related reference values of cholestanol and lathosterol concentrations, including also newborns and children.

Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3β,5α,6β-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3β-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3β,7α-diHCA. Moreover, 3β,7α-diHCA prevented the loss of motor neurons induced by 3β-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.

A new mechanism for formation of 7-ketocholesterol was recently described involving cytochrome P-450 (CYP)7A1-catalyzed conversion of 7-dehydrocholesterol into 7-ketocholesterol with cholesterol-7,8-epoxide as a side product. Some patients with cerebrotendinous xanthomatosis (CTX) and all patients with Smith-Lemli-Opitz syndrome (SLO) have markedly increased levels of 7-dehydrocholesterol in plasma and tissues. In addition, the former patients have markedly upregulated CYP7A1. We hypothesized that these patients may produce 7-ketocholesterol from 7-dehydrocholesterol with formation of cholesterol-7,8-epoxide as a side product. In accord with this hypothesis, two patients with CTX were found to have increased levels of 7-ketocholesterol and 7-dehydrocholesterol, as well as a significant level of cholesterol-7,8-epoxide. The latter steroid was not detectable in plasma from healthy volunteers. Downregulation of CYP7A1 activity by treatment with chenodeoxycholic acid reduced the levels of 7-ketocholesterol in parallel with decreased levels of 7-dehydrocholesterol and cholesterol-7,8-epoxide. Three patients with SLO were found to have markedly elevated levels of 7-ketocholesterol as well as high levels of cholesterol-7,8-epoxide. The results support the hypothesis that 7-dehydrocholesterol is a precursor to 7-ketocholesterol in SLO and some patients with CTX., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

Cerebrotendinous xanthomatosis (CTX) is a rare, difficult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX was compared with 5α-cholestanol. 7α,12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120-214 ng/ml) were ∼10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantification of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX by preventing morbidity and mortality.

A 34-year-old patient demonstrating pyramidal and cerebellar signs, accompanied by epilepsy, peripheral neuropathy, mental retardation and bilateral cataract was diagnosed with cerebrotendinous xanthomatosis based on the clinical picture, magnetic resonance imaging of the brain and serum sterol analysis. Tendon xanthomas were not observed in this case. After establishing the diagnosis, treatment with chenodeoxycholic acid and statin was introduced. During the next two years of the follow-up, serum cholestanol and 7α-hydroxycholesterol levels decreased in response to the therapy, but this was not reflected in the patient's neurological condition, which was slowly progressing. Treatment effectiveness in cerebrotendinous xanthomatosis is variable, notably better in patients who had started therapy before the injury to the nervous system took place. The present case report points to cerebrotendinous xanthomatosis as a rare cause of spinocerebellar syndrome, which might be treatable if diagnosed in early life.

Introduction: cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease caused by a deficiency of mitochondrial enzyme sterol 27-hydrolylase. Such a deficiency results in a reduced production of chenodeoxycholic acid and in an increased formation of cholestanol. It is clinically characterized by cataracts, diarrhoea, xanthomas, premature arteriosclerosis and a number of progressive neurological symptoms. Although cholestanol levels are used for the diagnosis of CTX, their correlation with the clinical symptoms and their prognostic usefulness have not been assessed so far., Methods: we reviewed 14 CTX patients diagnosed between 1995 and 2008 in two reference centres for the genetic diagnosis of this disorder, whose cholestanol levels had been recorded. We studied the main demographic, clinical and therapeutical data and their correlation with plasma cholestanol levels., Results: the average cholestanol level at diagnosis was 105.8 μmol/l. These levels did not correlate with any neurological symptoms or with disability at diagnosis scored by the EDSS. After treatment, all patients achieved a significant reduction in plasma cholestanol levels (average reduction of 91 μmol/l in an average follow-up of 34 months), although only one patient remained clinically stable., Conclusions: high cholestanol levels are very useful for diagnosis of CTX but they do not have a prognostic value (they do not correlate with severity). Normalisation of cholestanol levels is not always associated with clinical stabilisation. However, follow-up of cholestanol levels can be useful for the dose adjustment., (2010 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.)

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder believed to be exclusively caused by mutations in the CYP27A1 gene coding for the enzyme sterol 27-hydroxylase. Common findings in CTX are tendon xanthomas, cataracts and progressive neurological dysfunction. Here, we characterize an adult female patient with tendon xanthomas and classic biochemical findings of CTX (i.e. high levels of bile alcohols and cholestanol and extremely low levels of 27-hydroxycholesterol in plasma). Additionally, sterol 27-hydroxylase activity in cultured monocyte-derived macrophages from this patient was <5% of normal. Sequencing the CYP27A1 gene uncovered that the patient is heterozygous for two previously undescribed base substitutions in exon 8, C478A and C479A, which are expected to affect the haeme-binding domain of the enzyme. When expressed in HEK293 cells, the corresponding protein had only 8% of normal enzymatic activity. No other mutation was found in the open reading frame of the CYP27A1 gene, intron-exon boundaries or in the 5'-untranslated region up to 5000 bp distal to the translational start site. Sequencing mRNA isolated from leucocytes from the patient revealed a 1 : 1 ratio of mutated and nonmutated species, with total mRNA levels that were not significantly different from the controls. It is concluded that the patient is heterozygous for two mutations affecting one allele of the CYP27A1 gene and with at least one additional yet undefined gene that is of critical importance for the activity of sterol 27-hydroxylase.