Gas Chromatography and Flame-Ionization Detection of Non-Cholesterol Sterols as Indicators of Cholesterol Absorption and Synthesis in 158 Chinese Individuals with Normolipidemia, Hyperlipidemia, and Familial Hypercholesterolemia.

BACKGROUND There are limited studies on the effects of cholesterol homeostasis in populations at high risk for cardiovascular disease. We aimed to use gas chromatography and flame-ionization detection (GC-FID) of non-cholesterol sterols as indicators of cholesterol absorption and synthesis. Sterol indicators of cholesterol absorption included campesterol, stigmasterol, and sitosterol. Sterol indicators of cholesterol synthesis included squalene, 7-lathosterol, and desmosterol. MATERIAL AND METHODS A total of 158 participants were enrolled in 3 groups: healthy control (n=64), hyperlipidemia (n=69), and familial hypercholesterolemia (FH, n=25). Age, sex, blood pressure, blood glucose, and lipoprotein were collected, and cholesterol absorption and synthesis markers were determined by GC-FID. RESULTS All 6 cholesterol concentration indicators, except squalene, were significantly different among the 3 groups (all P<0.05); whereas in the ratio to cholesterol (%, sterols/cholesterol), only desmosterol and lathosterol were significantly different (P<0.05). Multifactorial regression analysis showed that triglycerides, total cholesterol, and desmosterol were independent risk factors affecting the development of hyperlipidemia (P<0.05). The efficacy of the ROC curve for the diagnosis of dyslipidemia was also higher for all 3 indices (Model 1, AUC=0.960). Model 1 was superior to Model 2 for the 6 indicators of cholesterol. For the FH and dyslipidemia groups, the 6-indicator model (Model 3) was shown to have a good diagnostic value (AUC=1.000). CONCLUSIONS The 6 sterol indicators of cholesterol absorption and synthesis had a dynamic course in all study participants. Desmosterol was an indicator of dyslipidemia. The combined use of the 6 sterol indicators differentiated between healthy individuals and patients with dyslipidemia and FH.

Effects of an 8-week aerobic exercise program on plasma markers for cholesterol absorption and synthesis in older overweight and obese men.

BACKGROUND: Increased physical activity is inversely related to the risk to develop cardiovascular disease (CVD). In a recent systematic review, it was reported that CVD patients had an increased cholesterol absorption and a decreased synthesis as compared with control participants. As increased physical activity levels reduce CVD risk, we hypothesized that exercise training will reduce cholesterol absorption and increase endogenous cholesterol synthesis in older overweight and obese men. METHODS: A randomized, controlled, crossover trial was performed. Seventeen apparently healthy older overweight and obese men were randomized to start with an aerobic exercise or no-exercise control period for 8 weeks, separated by 12 weeks washout. Fasting serum total cholesterol (TC) and non-cholesterol sterol concentrations were measured at baseline, and after 4 and 8 weeks. RESULTS: The aerobic exercise program did not affect serum TC concentrations. In addition, exercise did not affect TC-standardized serum concentrations of sitosterol and cholestanol that are markers for cholesterol absorption. However, a trend for reduced TC-standardized campesterol concentrations, which is another validated marker for cholesterol absorption, was observed as compared with control. Lathosterol concentrations, reflecting cholesterol synthesis, did not differ between both periods. CONCLUSIONS: Aerobic exercise training for 8 weeks did not lower serum TC concentrations in older overweight and obese men, but a trend towards a decrease in the cholesterol absorption marker campesterol was found. The cholesterol synthesis marker lathosterol did not change. TRIAL REGISTRATION: posted on www.clinicaltrials.gov as NCT03272061 on 7 September 2017.

Stocking density alters growth performance, serum biochemistry, digestive enzymes, immune response, and muscle quality of largemouth bass (Micropterus salmoides) in in-pond raceway system.

The effects of stocking density on growth performance, serum biochemistry, digestive enzymes, immune response, and muscle quality of largemouth bass (Micropterus salmoides) reared in nine in-pond raceway systems (IPRS, 22.0 m × 5.0 m × 2.0 m) were studied. M. salmoides with initial an body weight of 8.25 ± 0.51 g and body length of 6.99 ± 0.44 cm were reared at an initial stocking density of 90.91 ind./m3 (low stocking density, LSD), 113.63 ind./m3 (middle stocking density, MSD), and 136.36 ind./m3 (high stocking density, HSD) with triplication. After 300 days of culture, MSD recorded the highest final body weight, weight gain, specific growth rate, and yield, but the food conversion ratio in MSD was the lowest. The viscerosomatic index in LSD was significantly higher than other groups. The fish serum reared at HSD showed significantly lower total protein, higher total cholesterol, triglyceride, total bilirubin, glucose content, alanine transaminase, and aspartate transaminase activity. Significantly lower intestinal amylase, lipase, trypsin activities, hepatic superoxide dismutase (SOD) and catalase (CAT) activities, and higher malondialdehyde content were detected in HSD compared to others. The content of crude lipid, saturated fatty acid decreased, and total essential amino acid, delicious amino acid, and polyunsaturated fatty acid increased in muscle with stocking density increase. No significant difference was observed in muscle texture. Profitability analysis indicated the benefit-to-cost ratio varied between 1.10 and 1.68, of which MSD was significantly higher than others. The optimal stocking density for M. salmoides should be 113.63 ind./m3 in an IPRS farm.

Influence of pancreatic status on circulating plasma sterols in patients with cystic fibrosis.

Background Patients with cystic fibrosis (CF) have a reduced intestinal absorption of cholesterol and in a preliminary study we observed differences in plasma sterol profile between patients with pancreatic sufficiency (PS) and those with pancreatic insufficiency (PI). Therefore, we hypothesized that the sterol analysis may contribute to study the digestion and absorption state of lipids in patients with CF. To this aim we evaluated plasma sterols in a significant number of adult patients with CF in relation to the pancreatic status. Methods Beside cholesterol, we measured phytosterols and lathosterol as markers of intestinal absorption and hepatic biosynthesis, respectively, by gas-chromatography in plasma of adult CF patients with pancreatic sufficiency (PS-CF, n = 57), insufficiency (PI-CF, n = 97) and healthy subjects (control group, CT, n = 71). Results PI-CF patients had cholesterol and phytosterols levels significantly lower than PS-CF and CT (p < 5 × 10-10) suggesting a reduced intestinal absorption of sterols related to PI. Instead, lathosterol was significantly higher in PI-CF patients than PS-CF and CT (p < 0.0003) indicating an enhanced cholesterol biosynthesis. In PI-CF patients, phytosterols positively correlate with vitamin E (p = 0.004). Both the classes of molecules need cholesterol esterase for the intestinal digestion, thus the reduced levels of such lipids in serum from PI-CF patients may depend on a reduced enzyme activity, despite the pancreatic enzyme supplementation in all PI-CF patients. Conclusions A plasma sterols profile may be useful to evaluate the metabolic status of lipids in adult patients with CF and could help to manage the pancreatic enzyme supplementation therapy.

An Unbiased Lipid Phenotyping Approach To Study the Genetic Determinants of Lipids and Their Association with Coronary Heart Disease Risk Factors.

Direct infusion high-resolution mass spectrometry (DIHRMS) is a novel, high-throughput approach to rapidly and accurately profile hundreds of lipids in human serum without prior chromatography, facilitating in-depth lipid phenotyping for large epidemiological studies to reveal the detailed associations of individual lipids with coronary heart disease (CHD) risk factors. Intact lipid profiling by DIHRMS was performed on 5662 serum samples from healthy participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS). We developed a novel semi-targeted peak-picking algorithm to detect mass-to-charge ratios in positive and negative ionization modes. We analyzed lipid partial correlations, assessed the association of lipid principal components with established CHD risk factors and genetic variants, and examined differences between lipids for a common genetic polymorphism. The DIHRMS method provided information on 360 lipids (including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids), with a median coefficient of variation of 11.6% (range: 5.4-51.9). The lipids were highly correlated and exhibited a range of associations with clinical chemistry biomarkers and lifestyle factors. This platform can provide many novel insights into the effects of physiology and lifestyle on lipid metabolism, genetic determinants of lipids, and the relationship between individual lipids and CHD risk factors.

Relationship between the lipid composition of maternal plasma and infant plasma through breast milk.

INTRODUCTION: This study was motivated by the report that infant development correlates with particular lipids in infant plasma. OBJECTIVE: The hypothesis was that the abundance of these candidate biomarkers is influenced by the dietary intake of the infant. METHODS: A cohort of 30 exclusively-breastfeeding mother-infant pairs from a small region of West Africa was used for this observational study. Plasma and milk from the mother and plasma from her infant were collected within 24 h, 3 months post partum. The lipid, sterol and glyceride composition was surveyed using direct infusion MS in positive and negative ion modes. Analysis employed a combination of univariate and multivariate tests. RESULTS: The lipid profiles of mother and infant plasma samples are similar but distinguishable, and both are distinct from milk. Phosphatidylcholines (PC), cholesteryl esters (CEs) and cholesterol were more abundant in mothers with respect to their infants, e.g. PC(34:1) was 5.66% in mothers but 3.61% in infants (p = 3.60 × 10-10), CE(18:2) was 8.05% in mothers but 5.18% in infants (p = 1.37 × 10-11) whilst TGs were lower in mothers with respect to their infants, e.g. TG(52:2) was 2.74% in mothers and 4.23% in infants (p = 1.63 × 10-05). A latent structure model showed that four lipids in infant plasma previously shown to be biomarkers clustered with cholesteryl esters in the maternal circulation. CONCLUSION: This study found evidence that the abundance of individual lipid isoforms associated with infant development are associated with the abundance of individual molecular species in the mother's circulation.

Comparison of phytosterol intake from FFQ with repeated 24-h dietary recalls of the Adventist Health Study-2 calibration sub-study.

We evaluated the performance of an FFQ in estimating phytosterol intake against multiple 24-h dietary recalls (24HDR) using data from 1011 participants of the calibration sub-study of the Adventist Health Study-2 (AHS-2) cohort. Dietary assessments of phytosterol intake included a self-administered FFQ and six 24HDR and plasma sterols. Plasma sterols were determined using the GLC flame ionisation method. Validation of energy-adjusted phytosterol intake from the FFQ with 24HDR was conducted by calculating crude, unadjusted, partial and de-attenuated correlation coefficients (r) and cross-classification by race. On average, total phytosterol intake from the FFQ was 439·6 mg/d in blacks and 417·9 mg/d in whites. From the 24HDR, these were 295·6 mg/d in blacks and 351·4 mg/d in whites. Intake estimates of ß-sitosterol, stigmasterol, other plant sterols and total phytosterols from the FFQ had moderate to strong correlations with estimates from 24HDR (r 0·41-0·73). Correlations were slightly higher in whites (r 0·42-0·73) than in blacks (r 0·41-0·67). FFQ estimates were poorly correlated with plasma sterols as well as 24HDR v. plasma sterols. We conclude that the AHS-2 FFQ provided reasonable estimates of phytosterol intake and may be used in future studies relating phytosterol intake and disease outcomes.

Association between non-cholesterol sterol concentrations and Achilles tendon thickness in patients with genetic familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disorder that result in abnormally high low-density lipoprotein cholesterol levels, markedly increased risk of coronary heart disease (CHD) and tendon xanthomas (TX). However, the clinical expression is highly variable. TX are present in other metabolic diseases that associate increased sterol concentration. If non-cholesterol sterols are involved in the development of TX in FH has not been analyzed. METHODS: Clinical and biochemical characteristics, non-cholesterol sterols concentrations and Aquilles tendon thickness were determined in subjects with genetic FH with (n = 63) and without (n = 40) TX. Student-t test o Mann-Whitney test were used accordingly. Categorical variables were compared using a Chi square test. ANOVA and Kruskal-Wallis tests were performed to multiple independent variables comparison. Post hoc adjusted comparisons were performed with Bonferroni correction when applicable. Correlations of parameters in selected groups were calculated applying the non-parametric Spearman correlation procedure. To identify variables associated with Achilles tendon thickness changes, multiple linear regression were applied. RESULTS: Patients with TX presented higher concentrations of non-cholesterol sterols in plasma than patients without xanthomas (P = 0.006 and 0.034, respectively). Furthermore, there was a significant association between 5α-cholestanol, ß-sitosterol, desmosterol, 24S-hydroxycholesterol and 27-hydroxycholesterol concentrations and Achilles tendon thickness (p = 0.002, 0.012, 0.020, 0.045 and 0.040, respectively). CONCLUSIONS: Our results indicate that non-cholesterol sterol concentrations are associated with the presence of TX. Since cholesterol and non-cholesterol sterols are present in the same lipoproteins, further studies would be needed to elucidate their potential role in the development of TX.

Unravelling new pathways of sterol metabolism: lessons learned from in-born errors and cancer.

PURPOSE OF REVIEW: To update researchers of recently discovered metabolites of cholesterol and of its precursors and to suggest relevant metabolic pathways. RECENT FINDINGS: Patients suffering from inborn errors of sterol biosynthesis, transport and metabolism display unusual metabolic pathways, which may be major routes in the diseased state but minor in the healthy individual. Although quantitatively minor, these pathways may still be important in healthy individuals. Four inborn errors of metabolism, Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Niemann Pick disease types B (NPB) and C (NPC) result from mutations in different genes but can generate elevated levels of the same sterol metabolite, 7-oxocholesterol, in plasma. How this molecule is metabolized further is of great interest as its metabolites may have an important role in embryonic development. A second metabolite, abundant in NPC and NPB diseases, cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol), has recently been shown to be metabolized to the corresponding bile acid, 3ß,5α,6ß-trihydroxycholanoic acid, providing a diagnostic marker in plasma. The origin of cholestane-3ß,5α,6ß-triol is likely to be 3ß-hydroxycholestan-5,6-epoxide, which can alternatively be metabolized to the tumour suppressor dendrogenin A (DDA). In breast tumours, DDA levels are found to be decreased compared with normal tissues linking sterol metabolism to cancer. SUMMARY: Unusual sterol metabolites and pathways may not only provide markers of disease, but also clues towards cause and treatment.

A simple and precise method to detect sterol esterification activity of lecithin/cholesterol acyltransferase by high-performance liquid chromatography.

The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v/v) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride (P < 0.05), fractional esterification rate of HDL cholesterol (FERHDL) (P < 0.0001), and negatively correlated with apolipoprotein AI (apoAI) (P < 0.05) and HDL cholesterol (HDL-C) (P < 0.001). These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components and added substances, and will be a useful tool in the lipid metabolism study and the risk assessment of CVD.

A rare case of sterol-C4-methyl oxidase deficiency in a young Italian male: Biochemical and molecular characterization.

Inborn defects of cholesterol biosynthesis are metabolic disorders presenting with multi-organ and tissue anomalies. An autosomal recessive defect involving the demethylating enzyme C4-methyl sterol (SC4MOL) has been reported in only 4 patients so far. In infancy, all patients were affected by microcephaly, bilateral congenital cataracts, growth delay, psoriasiform dermatitis, immune dysfunction, and intellectual disability. Herein, we describe a new case of SC4MOL deficiency in which a 19-year-old Italian male was affected by bilateral congenital cataracts, growth delay and learning disabilities, behavioral disorders and small stature, but not microcephaly. Our patient had abundant scalp dandruff, without other skin manifestations. Analysis of the blood sterol profile showed accumulation of C4-monomethyl and C4-dimethyl sterols suggesting a deficiency of the SC4MOL enzyme. Sequencing of the MSMO1 gene (also known as the "SC4MOL" gene) confirmed mutations in each allele (c.731A>G, p.Y244C, which is already known, and c.605G>A, p.G202E, which is a novel variant). His father carried c.731A>G mutation, whereas his mother carried c.605G>A. Thus, the combination of multiple skills and methodologies, in particular, blood sterol profiling and genetic analysis, led to the diagnosis of a new case of a very rare defect of cholesterol biosynthesis. Consequently, we suggest that these two analyses should be performed as soon as possible in all undiagnosed patients affected by bilateral cataracts and developmental delay.

Metabonomic potential plasma biomarkers in abnormal fast buoyancy ascent escape-induced decompression sickness model and the protective effects of pyrrolidine dithiocarbamic acid.

BACKGROUND: Decompression sickness (DCS) induced by fast buoyancy ascent escape (FBAE) is a special DCS, characterized with cardiopulmonary injuries. Serum metabonomics of this type of DCS has not yet been studied. We proposed a metabonomics approach for assessing serum metabonomics changes and evaluating the preventive effect of pyrrolidine dithiocarbamic acid (PDTC) in FBAE-induced DCS rats. METHODS: Sixty-five (65) rats were divided into three groups, including the Control, DCS and PDTC groups. After receiving physiological saline or PDTC pretreatment, rats in the DCS and PDTC groups received the same protocol of simulated FBAE. Following this, a metabonomics approach - combined with pattern recognition methods including PCA and PLS-DA - was used to characterize the global serum metabolic profile on survival rats (five rats per group) associated with abnormal FBAE-induced DCS. As the VIP-value threshold cutoff of the metabolites was set to 2, metabolites above this threshold were filtered out as potential target biomarkers. RESULTS: Sixteen (16) distinct potential biomarkers in rat plasma were identified. PDTC significantly lowered DSC mortality from 60% to 10%, and alleviated ultrastructural alteration of the left ventricular apex compared to the DCS group. It was found that abnormal FBAE-induced DCS was closely related to disturbed fatty acid metabolism, glycerophospholipid metabolism, sterol lipid metabolism, and bile acid metabolism. With the presented metabonomic method, we systematically analyzed the protective effects of PDTC. CONCLUSION: The results demonstrated that PDTC administration could provide satisfactory effects on abnormal FBAE-induced DCS through partially regulating the perturbed metabolic pathways.

[The hypercholesterolemias in pregnancy: their etiology and diagnostic significance considerations]. / Hypercholesterolémie v tehotenství: úvahy o jejich etiologii a diagnostickém významu.

OBJECTIVE: Analysis of gestational hypercholesterolemia incidence in Prague population of healthy pregnant women. Diagnostic significance of non-cholesterol sterols as suitable markers of endogenous synthesis and intestinal absorption in etiology of gestational hypercholesterolemia. DESIGN: Retrospective study. PATIENTS AND METHODS: From 21 000 healthy pregnant women set of 84 patients with blood level of total cholesterol >7.0 mmol/l where noncholesterol sterols had been analyzed by use of GC/MS method on Finnigan MAT 120b. Lathosterol and desmosterol as markers of endogenous syntesis and campesterol and sitosterol as markers for intestinal absorbtion. Classical lipid parameters have been analyzed on Beckman Coulter and Cobas analyzators. RESULTS: The median of total cholesterol values in the set of 21 000 healthy pregnant women - 6,8 mmol/l was observed; median of LDLCh - 4.6 mmol/l and HDLCh - 2.2 mmol/l. The frequency of hypercholesterolemia values > 8.0 mmol/l 1:132 (!) was observed. The average values were for lathosterol 7.8 ± 1.7 µmol/l; desmosterol 4.7 ± 0.9 µmol/l; campesterol 9.8 ± 2.6 µmol/l; sitosterol 9.6 ± 3.8 µmol/l. The correlations of lathosterol with total cholesterol r = 0.524 as well as with non-HDLCh r = 0.35 and LDLCh r = 0.36 were observed. In campesterol or sitosterol as well as HDLCh or TAG no significant correlations have been observed. DISCUSSION: Pilot study for gestational hypercholesterolemias in Czech population of healthy pregnant women proved the high frequency of increased levels of total cholesterol (> 8.0 mmol/l) 1:132. Increased levels of lathosterol values could explain the hypercholesterolemia in pregnancy as result of higher endogene synthesis of cholesterol. CONCLUSION: Relatively high frequency of hypercholesterolemia in pregnancy is caused according to our findings by increased endogenous synthesis of cholesterol via lathosterol. The highly increased values of cholesterolemia during pregnancy could be efectivelly used for detection and after ending of lactation period for further differential diagnostic and treatment of previously undiagnosed familial hypercholesterolemias.

Lyso-glycosphingolipid abnormalities in different murine models of lysosomal storage disorders.

In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LC­MS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids. Specific elevations in tissue and plasma globotriaosylsphingosine were detected in α-galactosidase A-deficient mice; glucosylsphingosine in glucocerebrosidase-deficient mice and galactosylsphingosine in galactocerebrosidase-deficient animals. A similar investigation was conducted for two mouse models of Niemann Pick type C (Npc1nih and Npc1nmf164), revealing significant tissue elevation of several neutral glycosphingolipids and concomitant increased plasma glucosylsphingosine. This latter finding was recapitulated by analysis of plasma of NPC patients. The value of plasma glucosylsphingosine in biochemical confirmation of the diagnosis of NPC is discussed.

Bringing GC-MS profiling of steroids into clinical applications.

Abnormalities of steroid biosynthesis and excretion are responsible for the development and prevention of endocrine disorders, such as metabolic syndromes, cancers, and neurodegenerative diseases. Due to their biochemical roles in endocrine system, qualitative and quantitative analysis of steroid hormones in various biological specimens is needed to elucidate their altered expression. Mass spectrometry (MS)-based steroid profiling can reveal the states of metabolites in biological systems and provide comprehensive insights by allowing comparisons between metabolites present in cells, tissues, or organisms. In addition, the activities of many enzymes related to steroid metabolism often lead to hormonal imbalances that have serious consequences, and which are responsible for the progress of hormone-dependent diseases. In contrast to immunoaffinity-based enzyme assays, MS-based methods are more reproducible in quantification. In particular, high-resolution gas chromatographic (GC) separation of steroids with similar chemical structures can be achieved to provide rapid and reproducible results with excellent purification. GC-MS profiling therefore has been widely used for steroid analysis, and offers the basis for techniques that can be applied to large-scale clinical studies. Recent advances in analytical technologies combined with inter-disciplinary strategies, such as physiology and bioinformatics, will help in understanding the biochemical roles of steroid hormones. Therefore, comprehensive analytical protocols in steroid analysis for different research purposes may contribute to the elucidation of complex metabolic processes relevant to steroid function in many endocrine disorders, and in the identification of diagnostic biomarkers.

Reduced absorption and enhanced synthesis of cholesterol in patients with cystic fibrosis: a preliminary study of plasma sterols.

BACKGROUND: Low cholesterol is typically observed in the plasma of patients with cystic fibrosis (CF) contrasting with the subcellular accumulation of cholesterol demonstrated in CF cells and in mice models. However, the homeostasis of cholesterol has not been well investigated in patients with CF. METHODS: We studied the plasma of 26 patients with CF and 33 unaffected controls campesterol and ß-sitosterol as markers of intestinal absorption and lathosterol as a marker of de novo cholesterol biosynthesis by gas chromatography (GC-FID and GC-MS). RESULTS: Plasma campesterol and ß-sitosterol results were significantly (p=0.01) lower while plasma lathosterol was significantly higher (p=0.001) in patients with CF as compared to control subjects. Plasma cholesterol results were significantly lower (p=0.01) in CF patients. CONCLUSIONS: Our data suggest that the impaired intestinal absorption of exogenous sterols in patients with CF stimulates the endogenous synthesis of cholesterol, but the levels of total cholesterol in plasma remain lower. This may be due to the CFTR dysfunction that reduces cholesterol blood excretion causing the accumulation of cholesterol in liver cells and in other tissues contributing to trigger CF chronic inflammation.

Sitosterolemia Presenting as Pseudohomozygous Familial Hypercholesterolemia.

A young girl, age 8.5 years, presented with profound hypercholesterolemia and early xanthomatosis, suggesting homozygous familial (or type II) hypercholesterolemia. The patient's low density lipoprotein (LDL) receptor function and parental lipoprotein profiles were determined to be normal, prompting revision of the initial diagnosis to pseudohomozygous familial hypercholesterolemia. When she subsequently presented with giant platelets, the case was presented to colleagues on an electronic mailing list. It was recommended that plasma and sterol analysis be performed, which led to a diagnosis of sitosterolemia. The presentation of profound hypercholesterolomia in childhood that ultimately is not attributed as due to homozygous or compound heterozygous defects in the LDL receptor gene has been termed pseudohomozygous familial (or type II) hypercholesterolemia (PHT2HC). Patients diagnosed with PHT2HC subsequently confirmed to have sitosterolemia have been previously reported only rarely. The challenge of achieving accurate specific diagnosis and appropriate workup for these conditions in children is discussed in the context of this rare case and review of the historical literature concerning these conditions.

A Dietary Treatment Improves Cerebral Blood Flow and Brain Connectivity in Aging apoE4 Mice.

APOE ε4 (apoE4) polymorphism is the main genetic determinant of sporadic Alzheimer's disease (AD). A dietary approach (Fortasyn) including docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium has been proposed for dietary management of AD. We hypothesize that the diet could inhibit AD-like pathologies in apoE4 mice, specifically cerebrovascular and connectivity impairment. Moreover, we evaluated the diet effect on cerebral blood flow (CBF), functional connectivity (FC), gray/white matter integrity, and postsynaptic density in aging apoE4 mice. At 10-12 months, apoE4 mice did not display prominent pathological differences compared to wild-type (WT) mice. However, 16-18-month-old apoE4 mice revealed reduced CBF and accelerated synaptic loss. The diet increased cortical CBF and amount of synapses and improved white matter integrity and FC in both aging apoE4 and WT mice. We demonstrated that protective mechanisms on vascular and synapse health are enhanced by Fortasyn, independent of apoE genotype. We further showed the efficacy of a multimodal translational approach, including advanced MR neuroimaging, to study dietary intervention on brain structure and function in aging.

[Clinical Application of Methods for the Qualitative Evaluation of Lipid Abnormalities].

Recent lifestyle and social environment changes in Japan have been accompanied by increasing incidencerates of metabolic disorders, such as dyslipidemia and diabetes. Therefore, the rates of cardiovascular disease due to the progression of atherosclerosis are also increasing, and cardiovascular disease remains the leading cause of death in Japan. In particular, dyslipidemia, represented by hypercholesterolemia, hypertriglyceridemia, and hypoalphalipoproteinemia, is closely related to the onset and progression of atherosclerosis. Total cholesterol, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol have been used as quantitative markers of lipids to evaluate cardiovascular risks. However, these markers are not sufficient to fully assess the risks. Therefore, we focused on qualitative markers that represent lipid abnormalities, and examined the utility of qualitative lipids evaluation as clinical markers of atherosclerotic disorders. Previously, we reported that HDL and LDL subclasses and sterol markers are clinically important for evaluating the pathogenesis and risks of cardiovascular disorders. Moreover, lipoprotein subclasses may be useful as therapeutic markers for cardiovascular disorders, and oxysterols may also be useful as diagnostic markers for dementing disorders and diseases of the central nervous system. These issues remain to befully elucidated in the future.

Quantitative charge-tags for sterol and oxysterol analysis.

BACKGROUND: Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols. METHODS: Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3ß-hydroxy to 3-oxo groups. RESULTS: By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3ß-hydroxy group. Intra- and interassay CVs were <15%, and recoveries for representative oxysterols and cholestenoic acids were 85%-108%. By adopting a multiplex approach to isotope labeling, we analyzed up to 4 different samples in a single run. Using plasma samples, we could demonstrate the diagnosis of inborn errors of metabolism and also the export of oxysterols from brain via the jugular vein. CONCLUSIONS: This method allows the profiling of the widest range of sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism.