Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols 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 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.     

Differential effects of 24‐hydroxycholesterol and 27‐hydroxycholesterol on tyrosine hydroxylase and α‐synuclein in human neuroblastoma SH‐SY5Y cells

Evidence suggests that environmental and dietary factors may contribute to the pathogenesis of Parkinson’s disease (PD). High dietary intake of cholesterol is such a factor that has been shown to increase or decrease the risk of PD. However, because circulating cholesterol does not cross the blood–brain barrier, the mechanisms linking dietary cholesterol to the pathogenesis of PD remain to be understood. In contrast to cholesterol, the oxidized cholesterol metabolites (oxysterols), 24S‐hydroxycholesterol (24‐OHC) and 27‐hydroxycholesterol (27‐OHC), can cross the blood–brain barrier and may place the brain at risk of degeneration. In this study, we incubated the human neuroblastoma SH‐SY5Y cells for 24 h with 24‐OHC, 27‐OHC, or a mixture of 24‐OHC plus 27‐OHC, and have determined effects on tyrosine hydroxylase (the rate‐limiting enzyme in dopamine synthesis) levels, α‐synuclein levels, and apoptosis. We demonstrate that while 24‐OHC increases the levels of tyrosine hydroxylase, 27‐OHC increases levels of α‐synuclein, and induces apoptosis. Our findings show for the first time that oxysterols trigger changes in levels of proteins that are associated with the pathogenesis of PD. As steady state levels of 24‐OHC and 27‐OHC are tightly regulated in the brain, disturbances in these levels may contribute to the pathogenesis of PD.     

Cholesterol metabolism is associated with soluble amyloid precursor protein production in Alzheimer's disease

Disturbances of the cholesterol metabolism are associated with Alzheimer's disease (AD) risk and related cerebral pathology. Experimental studies found changing levels of cholesterol and its metabolites 24S‐hydroxycholesterol (24S‐OHC) and 27‐hydroxycholesterol (27‐OHC) to contribute to amyloidogenesis by increasing the production of soluble amyloid precursor protein (sAPP). The aim of this study was to evaluate the relationship between the CSF and circulating cholesterol 24S‐OHC and 27‐OHC, and the sAPP production as measured by CSF concentrations of sAPP forms in humans. The plasma and the CSF concentrations of cholesterol, 24S‐OHC and 27‐OHC, and the CSF concentrations of sAPPα, sAPPβ, and Aß1‐42 were assessed in subjects with AD and controls with normal cognition. In multivariate regression tests including age, gender, albumin ratio, and apolipoprotein E (APOE)ε4 status CSF cholesterol, 24S‐OHC, and 27‐OHC independently predicted the concentrations of sAPPα and sAPPβ. The associations remained significant when analyses were separately performed in the AD group. Furthermore, plasma 27‐OHC concentrations were associated with the CSF sAPP levels. The results suggest that high CSF concentrations of cholesterol, 24S‐OHC, and 27‐OHC are associated with increased production of both sAPP forms in AD.     

Biosynthesis of the regulatory oxysterol, 5-cholesten-3beta,25-diol 3-sulfate, in hepatocytes

Cellular cholesterol homeostasis is maintained through coordinated regulation of cholesterol synthesis, degradation, and secretion. Nuclear receptors for oxygenated cholesterol derivatives (oxysterols) are known to play key roles in the regulation of cholesterol homeostasis. We recently identified a sulfated oxysterol, 5-cholesten-3beta,25-diol 3-sulfate (25HC3S), that is localized to liver nuclei. The present study reports a biosynthetic pathway for 25HC3S in hepatocytes. Assays using mitochondria isolated from rats and sterol 27-hydroxylase (Cyp27A1) gene knockout mice indicated that 25-hydroxycholesterol (25HC) is synthesized by CYP27A1. Incubation of cholesterol or 25HC with mitochondrial and cytosolic fractions in the presence of 3'-phosphoadenosyl 5'-phosphosulfate resulted in the synthesis of 25HC3S. Real-time RT-PCR and Western blot analysis showed the presence of insulin-regulated hydroxycholesterol sulfotransferase 2B1b (SULT2B1b) in hepatocytes. 25HC3S, but not 25HC, decreased SULT2B1b mRNA and protein levels. Specific small interfering RNA decreased SULT2B1b mRNA, protein, and activity levels. These findings demonstrate that mitochondria synthesize 25HC, which is subsequently 3beta-sulfated to form 25HC3S.     

Up‐regulation of β‐amyloidogenesis in neuron‐like human cells by both 24‐ and 27‐hydroxycholesterol: protective effect of N‐acetyl‐cysteine

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27‐hydroxycholesterol (27‐OH) and 24‐hydroxycholesterol (24‐OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK‐N‐BE human neuroblastoma cells with patho‐physiologically relevant amounts of 27‐OH and 24‐OH showed that both oxysterols induce a net synthesis of Aβ_1‐42 by up‐regulating expression levels of amyloid precursor protein and β‐secretase, as well as the β‐secretase activity. Interestingly, cell pretreatment with N‐acetyl‐cysteine (NAC) fully prevented the enhancement of β‐amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β‐amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols‐induced Aβ toxic peptide accumulation in the brain.     

Analysis of bioactive oxysterols in newborn mouse brain by LC/MS

Unesterified cholesterol is a major component of plasma membranes. In the brain of the adult, it is mostly found in myelin sheaths, where it plays a major architectural role. In the newborn mouse, little myelination of neurons has occurred, and much of this sterol comprises a metabolically active pool. In the current study, we have accessed this metabolically active pool and, using LC/MS, have identified cholesterol precursors and metabolites. Although desmosterol and 24S-hydroxycholesterol represent the major precursor and metabolite, respectively, other steroids, including the oxysterols 22-oxocholesterol, 22R-hydroxycholesterol, 20R,22R-dihydroxycholesterol, and the C₂₁-neurosteroid progesterone, were identified. 24S,25-epoxycholesterol formed in parallel to cholesterol was also found to be a major sterol in newborn brain. Like 24S- and 22R-hydroxycholesterols, and also desmosterol, 24S,25-epoxycholesterol is a ligand to the liver X receptors, which are expressed in brain. The desmosterol metabolites (24Z),26-, (24E),26-, and 7α-hydroxydesmosterol were identified in brain for the first time     

24S-hydroxycholesterol alters activity of large-conductance Ca2+-dependent K+ (slo1 BK) channel through intercalation into plasma membrane

Oxysterols, oxidization products of cholesterol, are regarded as bioactive lipids affecting various physiological functions. However, little is known of their effects on ion channels. Using inside-out patch clamp recording, we found that naturally occurring side-chain oxidized oxysterols, 20S‑hydroxycholesterol, 22R‑hydroxycholesterol, 24S‑hydroxycholestero, 25‑hydroxycholesterol, and 27‑hydroxycholesterol, induced current reduction of large-conductance Ca²⁺- and voltage-activated K⁺ (slo1 BK) channels heterologously expressed in HEK293T cells. In contrast with side-chain oxidized oxysterols, naturally occurring ring oxidized ones, 7α‑hydroxycholesterol and 7‑ketocholesterol were without effect. By using 24S‑hydroxycholesterol (24S‑HC), the major brain oxysterol, we explored the inhibition mechanism. 24S‑HC inhibited Slo1 BK channels with an IC₅₀ of ~2 μM, and decreased macroscopic current by ~60%. This marked current decrease was accompanied by a rightward shift in the conductance-voltage relationship and a slowed activation kinetics, with the deactivation kinetics unaltered. Furthermore, the membrane sterol scavenger γ‑cyclodextrin was found to rescue slo1 BK channels from the inhibition, implicating that 24S-HC may be intercalated into the plasma membrane to affect the channel. These findings unveil a novel physiological importance of oxysterols from a new angle that involves ion channel regulation.     

Induction of apoptosis and necroptosis by 24(S)-hydroxycholesterol is dependent on activity of acyl-CoA:cholesterol acyltransferase 1

24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces necroptosis in human neuroblastoma SH-SY5Y cells. In the present study, we investigated the mechanisms by which 24S-OHC-induced cell death occurs. We found that lipid droplets formed at the early stages in the treatment of SH-SY5Y cells with 24S-OHC. These lipid droplets could be almost completely eliminated by treatment with a specific inhibitor or by siRNA knockdown of acyl-CoA:cholesterol acyltransferase 1 (ACAT1). In association with disappearance of lipid droplets, cell viability was recovered by treatment with the inhibitor or siRNA for ACAT1. Using gas chromatography–mass spectrometry, we confirmed that 24S-OHC-treated cells exhibited accumulation of 24S-OHC esters but not of cholesteryl esters and confirmed that accumulation of 24S-OHC esters was reduced when ACAT1 was inhibited. 24S-OHC induced apoptosis in T-lymphoma Jurkat cells, which endogenously expressed caspase-8, but did not induce apoptosis in SH-SY5Y cells, which expressed no caspase-8. In Jurkat cells treated with the pan-caspase inhibitor ZVAD and in caspase-8-deficient Jurkat cells, 24S-OHC was found to induce caspase-independent cell death, and this was partially but significantly inhibited by Necrostatin-1. Similarly, knockdown of receptor-interacting protein kinase 3, which is one of the essential kinases for necroptosis, significantly suppressed 24S-OHC-induced cell death in Jurkat cells treated with ZVAD. These results suggest that 24S-OHC can induce apoptosis or necroptosis, which of the two is induced being determined by caspase activity. Regardless of the presence or absence of ZVAD, 24S-OHC treatment induced the formation of lipid droplets and cell death in Jurkat cells, and this was suppressed by treatment with ACAT1 inhibitor. Collectively, these results suggest that it is ACAT1-catalyzed 24S-OHC esterification and the resulting lipid droplet formation that is the initial key event which is responsible for 24S-OHC-induced cell death.     

Assay of microsomal oxysterol 7α-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

A method of assaying hepatic cytochrome P-450, oxysterol 7α-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-³H]hydroxycholesterol as a substrate and hydroxypropyl-β-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7α,25-dihydroxycholesterol, into 3-oxo-7α,25-dihydroxy-4-cholesten by the activity of 3β-hydroxy-Δ⁵-C₂₇ steroid oxydoreductase, a microsomal NAD⁺ and NADP⁺ dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP⁺ into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7α-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (?33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (?30%) and also by the oxysterols 7α-hydroxycholesterol (?21%), 7β-hydroxycholesterol (?25%) and epicoprostanol (?20%), and by cyclosporin A (?26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.     

On the regulatory role of side-chain hydroxylated oxysterols in the brain. Lessons from CYP27A1 transgenic and Cyp27a1−/− mice

The two oxysterols, 27-hydroxycholesterol (27OH) and 24S-hydroxycholesterol (24OH), are both inhibitors of cholesterol synthesis and activators of the liver X receptor (LXR) in vitro. Their role as physiological regulators under in vivo conditions is controversial, however. In the present work, we utilized a previously described mouse model with overexpressed human sterol 27-hydroxylase (CYP27A1). The levels of 27OH were increased about 12-fold in the brain. The brain levels of HMG-CoA reductase mRNA and HMG-CoA synthase mRNA levels were increased. In accordance with increased cholesterol synthesis, most of the cholesterol precursors were also increased. The level of 24OH, the dominating oxysterol in the brain, was decreased by about 25%, most probably due to increased metabolism by CYP27A1. The LXR target genes were unaffected or slightly changed in a direction opposite to that expected for LXR activation. In the brain of Cyp27^−/− mice, cholesterol synthesis was slightly increased, with increased levels of cholesterol precursors but normal mRNA levels of HMG-CoA reductase and HMG-CoA synthase. The mRNA levels corresponding to LXR target genes were not affected. The results are consistent with the possibility that both 24OH and 27OH are physiological suppressors of cholesterol synthesis in the brain. The results do not support the contention that 27OH is a general activator of LXR target genes in this organ.     

Oxysterols and neurodegenerative diseases

In contrast to their parent molecule cholesterol, two of its side-chain oxidized metabolites are able to cross the blood–brain barrier. There is a concentration-driven flux of 24S-hydroxycholesterol (24S-OHC) from the brain into the circulation, which is of major importance for elimination of excess cholesterol from the brain. The opposite flux of 27-hydroxycholesterol (27-OHC) from the circulation into the brain may regulate a number of key enzymes within the brain. In vitro experiments suggest that the balance between the levels of these two molecules may be of importance for the generation of β-amyloid peptides. In primary cultures of rat hippocampal cells 27-OHC is able to suppress expression of the activity regulated cytoskeleton-associated protein (Arc), a protein important in memory consolidation which is reduced in patients with Alzheimer’s disease (AD). In the present work we explore the possibility that the flux of 27-OHC from the circulation into the brain represents the missing link between AD and hypercholesterolemia, and discuss the possibility that modification of this flux may be a therapeutic strategy. Lastly, we discuss the use of oxysterols as diagnostic markers in neurodegenerative disease.     

Increased maternal and fetal cholesterol efflux capacity and placental CYP27A1 expression in preeclampsia

Preeclampsia is a pregnancy-specific condition that leads to increased cardiovascular risk in later life. A decrease in cholesterol efflux capacity is linked to CVD. We hypothesized that in preeclampsia there would be a disruption of maternal/fetal plasma to efflux cholesterol, as well as differences in the concentrations of both placental sterol 27-hydroxylase (CYP27A1) and apoA1 binding protein (AIBP). Total, HDL-, and ABCA1-mediated cholesterol effluxes were performed with maternal and fetal plasma from women with preeclampsia and normotensive controls (both n = 17). apoA1 and apoE were quantified by chemiluminescence, and 27-hydroxycholesterol (27-OHC) by GC-MS. Immunohistochemistry was used to determine placental expression/localization of CYP27A1, AIBP, apoA1, apoE, and SRB1. Maternal and fetal total and HDL-mediated cholesterol efflux capacities were increased in preeclampsia (by 10–20%), but ABCA1-mediated efflux was decreased (by 20–35%; P < 0.05). Maternal and fetal apoE concentrations were higher in preeclampsia. Fetal plasma 27-OHC levels were decreased in preeclamptic samples (P< 0.05). Placental protein expression of both CYP27A1 and AIBP were localized around fetal vessels and significantly increased in preeclampsia (P = 0.04). Placental 27-OHC concentrations were also raised in preeclampsia (P < 0.05). Increased HDL-mediated cholesterol efflux capacity and placental CYP27A1/27-OHC could be a rescue mechanism in preeclampsia, to remove cholesterol from cells to limit lipid peroxidation and increase placental angiogenesis.     

Neuroprotective effects of blockers for T-type calcium channels

Background

Activation of the liver × receptors (LXRs) by exogenous ligands stimulates the degradation of β-amyloid 1–42 (Aβ42), a peptide that plays a central role in the pathogenesis of Alzheimer's disease (AD). The oxidized cholesterol products (oxysterols), 24-hydroxycholesterol (24-OHC) and 27-hydroxycholesterol (27-OHC), are endogenous activators of LXRs. However, the mechanisms by which these oxysterols may modulate Aβ42 levels are not well known.

Results

We determined the effect of 24-OHC and/or 27-OHC on Aβ generation in SH-SY5Y cells. We found that while 27-OHC increases levels of Aβ42, 24-OHC did not affect levels of this peptide. Increased Aβ42 levels with 27-OHC are associated with increased levels of β-amyloid precursor protein (APP) as well as β-secretase (BACE1), the enzyme that cleaves APP to yield Aβ. Unchanged Aβ42 levels with 24-OHC are associated with increased levels of sAPPα, suggesting that 24-OHC favors the processing of APP to the non-amyloidogenic pathway. Interestingly, 24-OHC, but not 27-OHC, increases levels of the ATP-binding cassette transporters, ABCA1 and ABCG1, which regulate cholesterol transport within and between cells.

Conclusion

These results suggest that cholesterol metabolites are linked to Aβ42 production. 24-OHC may favor the non-amyloidogenic pathway and 27-OHC may enhance production of Aβ42 by upregulating APP and BACE1. Regulation of 24-OHC: 27-OHC ratio could be an important strategy in controlling Aβ42 levels in AD.     

Oxidation of 7-dehydrocholesterol and desmosterol by human cytochrome P450 46A1

Cytochrome P450 (P450 or CYP) 46A1 is expressed in brain and has been characterized by its ability to oxidize cholesterol to 24S-hydroxycholesterol. In addition, the same enzyme is known to further oxidize 24S-hydroxycholesterol to the 24,25- and 24,27-dihydroxy products, as well as to catalyze side-chain oxidations of 7α-hydroxycholesterol and cholestanol. As precursors in the biosynthesis of cholesterol, 7-dehydrocholesterol has not been found to be a substrate of P450 46A1 and desmosterol has not been previously tested. However, 24-hydroxy-7-dehydrocholesterol was recently identified in brain tissues, which prompted us to reexamine this enzyme and its potential substrates. Here we report that P450 46A1 oxidizes 7-dehydrocholesterol to 24-hydroxy-7-dehydrocholesterol and 25-hydroxy-7-dehydrocholesterol, as confirmed by LC-MS and GC-MS. Overall, the catalytic rates of formation increased in the order of 24-hydroxy-7-dehydrocholesterol < 24-hydroxycholesterol < 25-hydroxy-7-dehydrocholesterol from their respective precursors, with a ratio of 1:2.5:5. In the case of desmosterol, epoxidation to 24S,25-epoxycholesterol and 27-hydroxylation was observed, at roughly equal rates. The formation of these oxysterols in the brain may be of relevance in Smith-Lemli-Opitz syndrome, desmosterolosis, and other relevant diseases, as well as in signal transduction by lipids.     

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48 h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

Oxysterols in the brain of the cholesterol 24-hydroxylase knockout mouse

Oxysterols are oxidised forms of cholesterol or its precursors. In this study we utilised the cholesterol 24-hydroxylase knockout mouse (Cyp46a1−/−) to study the sterol and oxysterol content of brain. Despite a great reduction in the abundance of 24S-hydroxycholesterol, the dominant metabolite of cholesterol in wild type brain, no other cholesterol metabolite was found to quantitatively replace this oxysterol in the Cyp46a1−/− mouse. Only minor amounts of other side-chain oxysterols including 22R-, 24R-, 25- and (25R),26-hydroxycholesterols were detected. In line with earlier studies, levels of cholesterol were similar in Cyp46a1−/− and wild type animals. However, the level of the cholesterol precursor, desomsterol, and its parallel metabolite formed via a shut of the mevalonate pathway, 24S,25-epoxycholesterol, were reduced in the Cyp46a1−/− mouse. The reduction in abundance of 24S,25-epoxycholesterol is interesting in light of a recent report indicating that this oxysterol promotes dopaminergic neurogenesis.     

7α-hydroxy-3-oxo-4-cholestenoic acid in cerebrospinal fluid reflects the integrity of the blood-brain barrier

There is a continuous flux of the oxysterol 27-hydroxycholesterol (27-OHC) from the circulation across the blood-brain barrier (BBB) into the brain. The major metabolite of 27-OHC in the brain is 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA). We confirm a recent report describing the presence of this metabolite in cerebrospinal fluid (CSF) at a relatively high concentration. A simple and accurate method was developed for assay of 7-HOCA in CSF based on isotope dilution-mass spectrometry and use of ²H₄-labeled internal standard. The concentration of this metabolite was found to be markedly increased in CSF from patients with a dysfunctional BBB. There was a high correlation between the levels of 7-HOCA in CSF and the CSF/serum albumin ratio. The concentration of 7-HOCA in CSF was not significantly affected by neurodegeneration. Our findings suggest that 7-HOCA could be used as a diagnostic marker for conditions with a dysfunctional BBB.