Evaluation of plasma cholestane-3β,5α,6β-triol and 7-ketocholesterol in inherited disorders related to cholesterol metabolism[S]

Oxysterols are intermediates of cholesterol metabolism and are generated from cholesterol via either enzymatic or nonenzymatic pathways under oxidative stress conditions. Cholestan-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7-KC) have been proposed as new biomarkers for the diagnosis of Niemann-Pick type C (NP-C) disease, representing an alternative tool to the invasive and time-consuming method of fibroblast filipin test. To test the efficacy of plasma oxysterol determination for the diagnosis of NP-C, we systematically screened oxysterol levels in patients affected by different inherited disorders related with cholesterol metabolism, which included Niemann-Pick type B (NP-B) disease, lysosomal acid lipase (LAL) deficiency, Smith-Lemli-Opitz syndrome (SLOS), congenital familial hypercholesterolemia (FH), and sitosterolemia (SITO). As expected, NP-C patients showed significant increase of both C-triol and 7-KC. Strong increase of both oxysterols was observed in NP-B and less pronounced in LAL deficiency. In SLOS, only 7-KC was markedly increased, whereas in both FH and in SITO, oxysterol concentrations were normal. Interestingly, in NP-C alone, we observed that plasma oxysterols correlate negatively with patient’s age and positively with serum total bilirubin, suggesting the potential relationship between oxysterol levels and hepatic disease status. Our results indicate that oxysterols are reliable and sensitive biomarkers of NP-C.

Ϫ 80°C, in 60 l aliquots, as plasma samples used for the oxysterol analysis are sensitive to thawing and stable for about 8 months at Ϫ 80°C (for ‫ف‬ 3 months at Ϫ 20°C). Briefl y, 5.0 l of 500 ng/ml 2 H 7 -C-triol and 2 H 7 -7KC standard solution, prepared in methanol, were mixed with 50 l of plasma sample in an Eppendorf tube. Liquid-liquid extraction was performed adding 500 l of ethyl acetate and mixing by vortex for 2 min. After centrifugation for 5 min at 14,000 rpm, the supernatant fl uid was transferred to a clean glass vial and dried under nitrogen steam. Derivatization was performed adding 20 l of derivatizing solution (100 mM dimethyl-aminobutyric acid imidazolide) at 65°C for 15 min. Finally, the sample was dried again and reconstituted with 200 l solvent, and 5 l was injected for the detection and quantifi cation of free or unesterifi ed oxysterols, C-triol, and 7-KC, as DMAB monoderivatives ( 6 ).

Patients and controls
We collected 51 samples obtained from 16 patients affected by NP-C, 5 samples from 2 NP-B patients, 6 samples from 2 patients affected by LAL defi ciency, 7 samples from 4 SLOS patients, 3 samples from 3 patients with FH, and 1 sample from 1 patient with SITO . All patients had biochemical, enzymatic and/or molecular diagnosis. Twelve out of 16 NP-C patients were those reported in the previous study ( 6 ), but, in the present study, for those patients, supplemental sample results were added. All patients were followed at the Division of Metabolism, "Bambino Gesù" Children's Hospital.
Blood samples were collected after obtaining informed consent during routine clinical evaluation following an overnight fast. The study was approved by Bambino Gesù Children's Hospital Ethics Committee (no. 1002_OPBG_2015). levels in a large series of CMRDs, including patients with NP-C and Niemann-Pick type B (NP-B) disease, lysosomal acid lipase (LAL) defi ciency, Smith-Lemli-Opitz syndrome (SLOS), congenital familial hypercholesterolemia (FH), and sitosterolemia (SITO).
Tandem mass spectrometry experiments were carried out on a 4000-QTrap mass spectrometer (ABSciex, Toronto, Ontario, Canada), equipped with a Turbo Ion Spray Source operating in positive ion mode. The monitored multiple reaction monitoring (MRM) transitions for C-triol-DMAB and 7-KC-DMAB derivatives were m/z 534/132 and 514/132, respectively.

Sample treatment procedure
Plasma was obtained from blood samples collected in EDTA K2 tubes immediately centrifuged for 5 min at 3,000 rpm. Plasma was separated from red cells and immediately stored frozen at A value of P < 0.001 was considered extremely statistically significant, a value of P from 0.001 to 0.01 was considered very statistically signifi cant, and a value of P from 0.01 to 0.05 was considered statistically signifi cant. C-triol and 7-KC values for control values were expressed as median and 2.5th to 97.5th percentiles of the distributions. Plasma C-triol and 7-KC values were expressed as median and minimum/maximum range of the distributions because of the small number of samples for each patient. Pearson coeffi cients were used to evaluate the relationship between continuous variables and the strength of their relationships. A value of P < 0.05 was considered statistically signifi cant.
Controls samples were obtained from 135 healthy agematched subjects. Child controls were subjects with normal neurological and liver function or subjects with traumatic injuries, and adults' plasma was obtained from healthy blood donors.

Statistical analysis
SPSS version 11.5.1 (SPSS Inc., Chicago, IL) was used as the statistical software. Descriptive statistics were presented as median. The preliminary Kolmogorov-Smirnov test was used to check variables that were under a normal distribution, to further use parametric or nonparametric tests. Statistically signifi cant differences between groups were analyzed using Student's t -test to Fig. 1. Levels of C-triol and 7-KC in patients affected by NP-C ( P < 0.0001), NP-B ( P < 0.0001), and LAL defi ciency ( P < 0.001). C-triol in SLOS patients was normal ( P > 0.05), whereas 7-KC was increased compared with control values ( P < 0.0001). Levels of C-triol and 7-KC in patients affected by FH and SITO were normal ( P > 0.05). FH patients plasma oxysterols were within the control range ( Table 1 ). Figure 1 shows the relative concentrations of the two oxysterols in CMRDs in comparison with controls.
Although the oxysterols are known to be cholesterol oxidation products, no correlations were found between the plasma concentrations of the two oxysterols in CMRDs with the levels of t-chol, LDL, or HDL when analyzed in the same sample (Pearson <0.3, P > 0.01) (supplementary Table 3). Indeed, oxysterols in CMRDs were not correlated with t-bil or with age. Remarkably, in NP-C patients alone we found signifi cant correlations between the plasma concentrations of oxysterols and both t-bil and age, as shown in Table 2 , Fig. 2 , and Fig. 3 .

DISCUSSION
The conversion of cholesterol to oxysterols occurs by enzymatic and nonenzymatic reactions. The nonenzymatic reactions mainly affect the sterol ring, whereas the enzymatic ones react in the side chain of sterol structures by enzymatic cholesterol hydroxylation, catalyzed by different types of hydroxylases. However, some exceptions exist: 25-hydroxycholesterol and 7 ␣ -hydroxy-cholesterol can be generated by either mechanism ( 7 ). Oxysterol production from nonenzymatic reactions is not controlled by enzymatic mechanisms, so their formation depends on the concentration of reactive oxygen species and the structure of the target lipids to be oxidized ( 8 ). The increase of plasma C-triol and 7-KC observed in NP-C disease is the consequence of the nonenzymatic cholesterol oxidation promoted by oxidative stress, as reported in human NP-C mutant fi broblasts ( 9, 10 ), in tissues from NPC1 mutant mice ( 11 ), and in NP-C patients ( 12 ). The defect of NPC1 or NPC2 transporters, occurring in NP-C disease, makes available for the various cell functions a very small amount of free cholesterol from lysosomes, causing an impaired lipid traffi cking and cholesterol synthesis upregulation ( 13 ). Subsequently, free cholesterol accumulation leads to high oxysterol generation. In particular, C-triol is produced by epimeric 5,6-epoxides, whereas 7-KC is produced

Biological variation of C-triol and 7-KC in plasma
We measured C-triol and 7-KC concentrations in plasma samples of 135 healthy subjects from 0.2 to 38 years old, the age range that covers the ages of our patients. C-triol control values were median 4.1 ng/ml and 2.5th to 97.5th percentile 1.1-21.9 ng/ml, respectively; 7-KC control values were median 16.1 ng/ml and 2.5th to 97.5th percentile 3.8-39.8 ng/ml, respectively.
Data showed a signifi cant direct correlation between C-triol and 7-KC (Pearson 0.404, P < 0.001), whereas their values were not correlated with age.

Patients' results
Overall, the 16 NP-C patients showed a signifi cant increase of both oxysterols: C-triol median value was 55.3 ng/ml (range 16.3-608, P < 0.001), and the 7-KC median value was 86.0 ng/ml (range 21.9-963, P < 0.001). Only in 1 out of 51 NP-C samples did we fi nd a C-triol value within the control range, whereas in 4 out of 51 samples, 7-KC plasma concentrations were found to be normal ( Table 1 ).
Despite a massive increase of total plasma cholesterol (median 336 mg/dl, control values 120-200), in the three "." indicates that the signifi cance of the correlation between one parameter and itself is maximum (unquantifi able number<0.00001). a P < 0.01. b P < 0.05.
presenting initially with severe cholestasis and showing with age a parallel decline of oxysterols and total bilirubin. Besides NP-C disease, two recent studies, based on the determination of 7-KC alone, showed its increase in NP-B disease and in SLOS, two inherited disorders due to deficient activity of the lysosmal enzyme sphingomyelinase and of 7-dehydrocholesterol reductase, respectively ( 15,16 ). A further study, showed that both 7-KC and C-triol were elevated in NP-B patients ( 14 ). A very recent study described high levels of C-triol in NP-C, cerebrotendinous xanthomatosis, and LAL defi ciency ( 17 ).
In NP-B disease, the defi cient activity of acid sphingomyelinase impairs the conversion of SM to ceramide, which causes massive intracellular accumulation of SM. Interestingly, a recent study showed that the free cholesterol transporter NPC2 is inhibited by SM excess and upregulated by ceramide, indicating that sphingomyelinase also participates in the complex machinery that regulates the secretion of by an attack of cholesterol by oxidative radical species resulting in the formation of 7-hydroperoxy-cholesterol, which decomposes to give either 7-KC or 7 ␣ -or 7-␤ -hydroxycholesterol, under increased cellular oxidative stress conditions. The current results confi rmed that C-triol has a higher sensitivity in detecting NP-C patients when compared with 7-KC ( Fig. 1 ) ( 6, 14 ). Remarkably, in NP-C patients we observed two novel signifi cant correlations between the plasma oxysterols and total bilirubin and patients' age. This seems to indicate that in NP-C the elevation of oxysterols refl ects liver involvement, following different stages of the disease ( 13 ). It is well known that disease phenotype changes according to the patient's age, ranging from a picture dominated by visceral (i.e., hepatic) manifestations in the early onset cases, moving to a "pure" (nonhepatic) neurological phenotype in adulthood. Following these correlations, it was possible to track the hepatic disease course in two early onset infants both in NP-B patients are likely to refl ect the lysosomal free cholesterol accumulation with a mechanism similar to NP-C.
We found that plasma oxysterol concentrations were also elevated in LAL defi ciency patients, however, to a lesser extent when compared with NP-C or NP-B. The fi nding of increased C-triol and 7-KC in LAL defect is diffi cult to explain because the intracellular free cholesterol concentration would presumably be reduced in LAL deficiency. Following the receptor-mediated cell endocytosis of LDL, the hydrolysis of LDL-derived cholesteryl esters is catalyzed in the lysosome by LAL. Therefore, a defi ciency of this enzyme would result in the lysosmal accumulation of esterifi ed cholesterol and in a relative depletion of free cholesterol, which may likely cause the activation of endogenous cholesterol biosynthesis by the upregulation of HMG-CoA reductase, resulting in the characteristic hypercholesterolemia observed in these patients and contributing to the formation of oxysterols ( 20,21 ). free cholesterol from the lysosomal compartment ( 18 ). The effects of SM excess on cellular cholesterol transport have been demonstrated in NP-B fi broblasts ( 19 ). By confocal microscopy and cellular lipid mass measurement, this study showed the lysosomal cosequestration and trapping of SM and free cholesterol in mutant NP-B cell lines. Furthermore, NP-B fi broblasts showed also a signifi cant increase of de novo cholesterol biosynthesis when compared with control fi broblast. These results demonstrate that free cholesterol accumulation in lysosomes, caused by an impairment of its transport out of the organelle, upregulates the endogenous cholesterol synthesis, which further increases the cellular storage. Interestingly, a recent study showed that cholesterol transfer mediated by NPC2 protein is inhibited by SM excess, indicating that sphingomyelinase participates in the complex machinery that regulates the secretion of free cholesterol from the lysosomal compartment ( 19 ). Therefore, the high oxysterol levels observed In SLOS patients, the defect of 7-dehydrocholesterol reductase causes a massive accumulation of 7-dehydrocholesterol (7-DHC) ( 22 ). The fi nding of increased 7-KC alone in SLOS patients can therefore be easily explained by conversion of excessive 7-DHC into 7-KC ( 23 ).
FH is a rare autosomal dominant genetic disorder that leads to a massive increase of blood cholesterol in most cases due to mutations in the LDL receptor gene ( 24 ). In FH, the LDL particles cannot cross the cellular membrane, resulting in the accumulation of cholesterol in the blood compartment with relatively low free cholesterol concentrations in the intracellular compartment. Our fi nding of normal oxysterol levels in FH is consistent with this mechanism.
Although the processes that regulate cholesterol metabolism within cellular and subcellular compartment cells still remains to be fully elucidated, data obtained in the present study may still help to clarify some of the mechanisms that underlie the regulation of cellular cholesterol machinery.
Our fi ndings confi rm that plasma oxysterols are reliable and sensitive biomarkers of NP-C disease, allowing a faster diagnosis than with the more invasive "Filipin test." However, a high concentration of plasma oxysterols is not specifi c for NP-C disease as we found increased levels also in other CMRDs. A careful clinical evaluation is needed to distinguish NP-C disease from other CMRDs that present an increase of both plasma oxysterols.