Case Report: Early Treatment With Chenodeoxycholic Acid in Cerebrotendinous Xanthomatosis Presenting as Neonatal Cholestasis.

Background: Cerebrotendinous xanthomatosis (CTX) is an inborn disorder of bile acid synthesis which causes progressive accumulation of toxic metabolites in various organs, particularly in brain and tendons. Most cases are diagnosed and treated in the second or third decade of life, when neurological involvement appears. We describe a case of CTX presenting as neonatal cholestasis. Results: The child presented cholestasis at 2 months of life. In the following months jaundice slowly disappeared, with a normalization of bilirubin and aminotransferases, respectively, at 6 and 8 months. A LC-Mass Spectrometry of the urines showed the presence of cholestanepentols glucuronide, which led to the suspicion of cerebrotendinous xanthomatosis. The diagnosis was confirmed by the dosage of cholestanol in serum and the molecular genetic analysis of the CYP27A1 gene. Therapy with chenodeoxycholic acid (CDCA) was started at 8 months and is still ongoing. The child was monitored for 13 years by dosage of serum cholestanol and urinary cholestanepentols. A strictly biochemical and neurological follow up was performed and no sign of neurological impairment was observed. Conclusions: Prompt diagnosis and treatment of CTX presenting as neonatal cholestasis may prevent further neurological impairment.

Correction to: Nutraceutical approach for the management of cardiovascular risk - a combination containing the probiotic Bifidobacterium longum BB536 and red yeast rice extract: results from a randomized, double-blind, placebo-controlled study.

Following publication of the original article [1], the authors reported an error in the affiliation of the third author, Sara Gandini. The correct affiliation should read: Division of Epidemiology and Biostatistics, IEO, European Institute of Oncology IRCCS, Milan, Italy.

Nutraceutical approach for the management of cardiovascular risk - a combination containing the probiotic Bifidobacterium longum BB536 and red yeast rice extract: results from a randomized, double-blind, placebo-controlled study.

BACKGROUND: Probiotics incorporated into dairy products have been shown to reduce total (TC) and LDL cholesterolemia (LDL-C) in subjects with moderate hypercholesterolemia. More specifically, probiotics with high biliary salt hydrolase activity, e.g. Bifidobacterium longum BB536, may decrease TC and LDL-C by lowering intestinal cholesterol reabsorption and, combined with other nutraceuticals, may be useful to manage hypercholesterolemia in subjects with low cardiovascular (CV) risk. This study was conducted to evaluate the efficacy and safety of a nutraceutical combination containing Bifidobacterium longum BB536, red yeast rice (RYR) extract (10 mg/day monacolin K), niacin, coenzyme Q10 (Lactoflorene Colesterolo®). The end-points were changes of lipid CV risk markers (LDL-C, TC, non-HDL-cholesterol (HDL-C), triglycerides (TG), apolipoprotein B (ApoB), HDL-C, apolipoprotein AI (ApoAI), lipoprotein(a) (Lp(a), proprotein convertase subtilisin/kexin type 9 (PCSK9)), and of markers of cholesterol synthesis/absorption. METHODS: A 12-week randomized, parallel, double-blind, placebo-controlled study. Thirty-three subjects (18-70 years) in primary CV prevention and low CV risk (SCORE: 0-1% in 24 and 2-4% in 9 subjects; LDL-C: 130-200 mg/dL) were randomly allocated to either nutraceutical (N = 16) or placebo (N = 17). RESULTS: Twelve-week treatment with the nutraceutical combination, compared to placebo, significantly reduced TC (- 16.7%), LDL-C (- 25.7%), non-HDL-C (- 24%) (all p < 0.0001), apoB (- 17%, p = 0.003). TG, HDL-C, apoAI, Lp(a), PCSK9 were unchanged. Lathosterol:TC ratio was significantly reduced by the nutraceutical combination, while campesterol:TC ratio and sitosterol:TC ratio did not change, suggesting reduction of synthesis without increased absorption of cholesterol. No adverse effects and a 97% compliance were observed. CONCLUSIONS: A 12-week treatment with a nutraceutical combination containing the probiotic Bifidobacterium longum BB536 and RYR extract significantly improved the atherogenic lipid profile and was well tolerated by low CV risk subjects. TRIAL REGISTRATION: NCT02689934 .

Proteomics of liquid biopsies: Depicting RCC infiltration into the renal vein by MS analysis of urine and plasma.

Liquid biopsies, as blood and urine, could offer an invaluable, easily accessible source of biomarkers, and evidences for elucidating the pathological processes. Only few studies integrated the proteomes driven by more than one biofluid. Furthermore, it is not clear which biofluid better mirrors the alterations triggered by disease. Venous infiltrating RCC(Renal Cell Carcinoma) could represent an advantageous model for exploring this aspect. Herein, we investigate how blood and urine "proteomically" reflect the changes occurring during RCC infiltration into renal vein(RV) by label-free nLC-ESI-MS/MS. We found 574 and 58 differentially expressed proteins(DEPs) in response to vascular involvement. To the augment of vascular involvement, the abundance of only three proteins in urine(UROM,RALA,CNDP1) and two in plasma(APOA1,K2C1) diminished while increased for twenty-six urinary proteins. 80 proteins were found both in urine and plasma, among which twenty-eight were DEPs. A huge overlap between the two biofluids was highlighted, as expected, being urine the filtrate of blood. However, this consistency decreases when RV-occlusion occurs suggesting alternative protein releases, and a loss of kidney architecture. Moreover, several proteomic and functional signatures were biofluid-specific. In conclusion, the complementarity between the specimens allowed to achieve a deeper level of molecular complexity of the RCC venous infiltration. SIGNIFICANCE: Although plasma and urine are strongly interconnected, only few proteomic studies investigated the complementarity of these fluids as bio-sources of information. Moreover, none of them was focused to their analysis and comparison in the context of vascular infiltration of renal cancer. Herein, new insights were gained regarding the impact into urinary and plasma proteome of the changes triggered by the ccRCC invasion into vascular system and renal vein. Furthermore, the integration of the information driven by the two liquid biopsies permits to unravel biological processes otherwise lost.

Histology-guided proteomic analysis to investigate the molecular profiles of clear cell Renal Cell Carcinoma grades.

Renal Cell Carcinoma (RCC) is the most frequent form of kidney cancer and approximately 80% of cases are defined as clear cell RCC (ccRCC). Among the histopathological factors, tumour grade represents one of the most important parameters to evaluate ccRCC progression. Nonetheless, the molecular processes associated with the grading classification haven't been deeply investigated thus far. Therefore, the aim of this study was to uncover protein alterations associated with different ccRCC grade lesions. Formalin-fixed paraffin-embedded samples from ccRCC patients were analysed by histology-guided MALDI-MSI and shotgun proteomics in order to study the biological processes implicated in ccRCC. MALDI-MSI data highlighted signals able to discriminate among different grades (AUC > 0.8). The ion at m/z 1428.92 was identified as Vimentin and was overexpressed in grade 4 lesions, whereas ions at m/z 944.71, m/z 1032.78 and m/z 1325,99 were identified as histones H2A, H3, and H4, respectively. nLC-ESI-MS/MS analysis provided a further list of proteins and their abundances, showing a difference in protein content among the four grades. Moreover, the obtained molecular profiles showed a correspondence with the different Cancer-Specific Survival rate at 10 years post-surgery, as reported in literature. SIGNIFICANCE: Despite the generally accepted role of tumour grade in ccRCC diagnosis, the proteomic processes associated with the different tumour grades has not been extensively studied and doing so may provide insights into the development of the disease. In the current study, data obtained using MALDI-MSI was integrated with that obtained using nLC-ESI-MS/MS to highlight the proteomic alterations underlying the different ccRCC grades. The combined approach identified vimentin and three histones (H2A, H3 and H4) that were able to discriminate among the four grades whilst the nLC-ESI-MS/MS analysis alone provided a further list of proteins with an altered abundance. Furthermore, there was a good correlation between the molecular profiles generated for each grade and the different Cancer-Specific Survival rate at 10 years post-surgery. Such findings could be a valuable starting point for further studies aimed at clarifying the molecular events that occur during the development of ccRCC.

Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging in the Study of Gastric Cancer: A Mini Review.

Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide and the disease outcome commonly depends upon the tumour stage at the time of diagnosis. However, this cancer can often be asymptomatic during the early stages and remain undetected until the later stages of tumour development, having a significant impact on patient prognosis. However, our comprehension of the mechanisms underlying the development of gastric malignancies is still lacking. For these reasons, the search for new diagnostic and prognostic markers for gastric cancer is an ongoing pursuit. Modern mass spectrometry imaging (MSI) techniques, in particular matrix-assisted laser desorption/ionisation (MALDI), have emerged as a plausible tool in clinical pathology as a whole. More specifically, MALDI-MSI is being increasingly employed in the study of gastric cancer and has already elucidated some important disease checkpoints that may help us to better understand the molecular mechanisms underpinning this aggressive cancer. Here we report the state of the art of MALDI-MSI approaches, ranging from sample preparation to statistical analysis, and provide a complete review of the key findings that have been reported in the literature thus far.

Evaluation of cholesterol metabolism in cerebrotendinous xanthomatosis.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is a treatable bile acid disorder caused by mutations of CYP27A1. The pathogenesis of neurological damage has not been completely explained. Oral chenodeoxycholic acid (CDCA) can lead to clinical stabilization, but in a subgroup of patients the disease progresses despite treatment. In the present study, we aimed at clarifying cholesterol metabolism abnormalities and their response to CDCA treatment, in order to identify reliable diagnostic and prognostic markers and understand if differences exist between stable patients and those with neurological progression. METHODS: We enrolled 19 untreated CTX patients and assessed serum profile of bile acids intermediates, oxysterols, cholesterol, lathosterol, and plant sterols. Then we performed a long-term follow up during CDCA therapy, and compared biochemical data with neurological outcome. RESULTS: We observed increase of cholestanol, 7α-hydroxy-4-cholesten-3-one (7αC4), lathosterol, and plant sterols, whereas 27-hydroxycholesterol (27-OHC) was extremely low or absent. CDCA treatment at a daily dose of 750 mg normalized all biochemical parameters except for 7αC4 which persisted slightly higher than normal in most patients, and 27-OHC which was not modified by therapy. Biochemical evaluation did not reveal significant differences between stable and worsening patients. DISCUSSION: Cholestanol and 7αC4 represent important markers for CTX diagnosis and monitoring of therapy. Treatment with CDCA should aim at normalizing serum 7αC4 as well as cholestanol, since 7αC4 better mirrors 7α-hydroxylation rate and is thought to be correlated with cholestanol accumulation in the brain. Assessment of serum 27-OHC is a very good tool for biochemical diagnosis at any stage of disease. Lathosterol and plant sterols should be considered as additional markers for diagnosis and monitoring of therapy. Further studies including long-term assessment of bile acid intermediates in cerebrospinal fluid are needed in patients who show clinical progression despite treatment.

Duodenal diverted sleeve gastrectomy with ileal interposition does not cause biliary salt malabsorption.

BACKGROUND: Duodenal diverted sleeve gastrectomy with ileal interposition (DDSG-II) is a bariatric-metabolic operation designed to treat type 2 diabetes mellitus (T2DM). It is restrictive (SG) but also acts on the small bowel with functional effects. The objective of the present study was to investigate whether or not it is also a malabsorptive operation. METHODS: Twelve obese patients (9 female and 3 male) affected by T2DM had DDSG-II. Follow-up was every 3 months, and the results after 1 year are reported here. Clinical conditions, related to diabetes and malnutrition, changes in weight, body mass index, fasting glucose plasma levels, HbA1c (glycated hemoglobin %), basal insulin, vitamin B12, folic acid, vitamin D, total proteins, albumin, and hemoglobin were recorded. Basal plasma levels of FGF19 (pg/mL) and of 7α-hydroxy-4-cholesten-3-one (C4) (µg/dL) were also determined for the diagnosis of biliary salt malabsorption. The results were expressed as mean±SEM, and the differences between times compared by the Mann Whitney U test; P<.05 was considered significant. RESULTS: After 1 year, all patients had a significant weight loss (-33.2±3 kg) with T2DM remission according to the American Diabetes Association criteria (11), criteria. No significant changes in total proteins, albumin, hemoglobin, and vitamins (B6, B12, and D) were detected. C4 did not change after the operation (2±.5 µg/dL versus 1.6±.5 µg/dL), whereas FGF19 significantly increased (from 85±11.2 pg/mL to 166.4±28.2 pg/mL, P<.04). Only 2 patients had mild symptoms of malabsorption. CONCLUSIONS: DDSG-II is effective for treatment of T2DM obese patients, increases the enterokine FGF19, and does not cause biliary salt malabsorption.

Age-associated alterations in cholesterol homeostasis: evidence from a cross-sectional study in a Northern Italy population.

BACKGROUND: The modifications of cholesterol metabolism associated with aging are ill-defined. The objective of this study was to define age-associated alterations of the different metabolic pathways controlling cholesterol homeostasis by analyzing circulating sterols. METHODS: We analyzed serum samples collected from 201 adult (75 male, 126 female) subjects within the epidemiological MICOL study (Multicentrica Italiana Colelitiasi). The age range was 38-79 years; 103 had evidence of gallstones. The concentrations of the different sterols, recognized as markers of the main pathways of cholesterol homeostasis, were analyzed by gas chromatography-mass spectrometry, including lathosterol (synthesis), campesterol and sitosterol (absorption), and 7α-hydroxy-4-cholesten-3-one (degradation to bile acids). RESULTS: A significant direct correlation was detected between age and cholesterol levels (r =0.34, P<0.01). The lathosterol/cholesterol ratio was lower in older age quartiles (P<0.05 by analysis of variance), with an inverse correlation between the lathosterol/cholesterol ratio and age (r=-0.32, P<0.01). Such correlation was particularly evident in females. The campesterol/cholesterol and sitosterol/cholesterol ratios were inversely correlated with aging in control, but not in gallstone patients. The levels of 7α-hydroxy-4-cholesten-3-one were not correlated with age. CONCLUSION: These data show a reduction of cholesterol synthesis with aging which is associated with increased circulating cholesterol levels. The finding might be related to a reduced metabolic need for cholesterol in advancing age, leading to a downregulation of the main mechanisms of cholesterol intake in the liver. A different age-related behavior was observed in gallstone-free versus gallstone patients regarding cholesterol absorption. The possible implications in terms of the pharmacological management of hypercholesterolemia in the elderly remain to be defined.

In vivo degradation of cholesterol to bile acids is reduced in patients receiving parenteral nutrition.

BACKGROUND: Artificial nutrition is frequently associated with hepatobiliary complications, probably due to the inherent derangement of the gastrointestinal tract physiology. Alterations of hepatic lipid metabolism are likely to be involved. The aim of the present study was to investigate the effect of artificial nutrition on bile acid production, a key event in cholesterol homeostasis, in humans. PATIENTS AND METHODS: Eleven patients receiving artificial nutrition, either parenteral nutrition (PN; n = 6) or enteral nutrition (EN; n = 5) with no previous history of liver disease, underwent analysis of cholesterol 7α-hydroxylation rates in vivo, a measure of bile acid formation, by isotope release analysis after intravenous injection of [7α-(3)H]cholesterol. The results were compared with those obtained in a population of 16 age-matched control subjects. RESULTS: Hydroxylation rates were lower in patients with artificial nutrition (PN: 94 ± 13 mg/d; EN: 230 ± 39 mg/d, mean ± SEM) when compared with controls (385 ± 47 mg/d) (P < .01, 1-way analysis of variance). In a patient receiving EN, hydroxylation rates increased 3.5-fold after treatment with the cholecystokinin analogue ceruletide (20 µg bid for 2 weeks intramuscularly). Serum lathosterol-to-cholesterol ratio, a marker of cholesterol synthesis, was also significantly reduced in artificial nutrition, whereas serum levels of fibroblast growth factor 19 (FGF19) were increased. CONCLUSION: In vivo 7α-hydroxylation is suppressed in artificial nutrition, particularly in PN. The finding associates with reduced cholesterol production, possibly as a metabolic consequence. The data suggest a regulatory role of gastrointestinal hormones and FGF19 on bile acid production and might suggest a pathophysiological basis for some common complications of artificial nutrition, such as gallstone disease and cholestasis.

Cholesterol metabolism after bariatric surgery in grade 3 obesity: differences between malabsorptive and restrictive procedures.

OBJECTIVE: Malabsorptive bariatric surgery (biliopancreatic diversion and biliointestinal bypass [BIBP]) reduces serum cholesterol levels more than restrictive surgery (adjustable gastric banding [AGB]), and this is thought to be due to greater weight loss. Our aim was to evaluate the changes of cholesterol metabolism induced by malabsorptive and restrictive surgery independent of weight loss. RESEARCH DESIGN AND METHODS: In a nonrandomized, self-selected, unblinded, active-comparator, bicenter, 6-month study, glucose metabolism (blood glucose and serum insulin levels and homeostasis model assessment of insulin resistance [HOMA-IR] index) and cholesterol metabolism (absorption: serum campesterol and sitosterol levels; synthesis: serum lathosterol levels; catabolism: rate of appearance and serum concentrations of serum 7-α- and serum 27-OH-cholesterol after infusions of deuterated 7-α- and 27-OH-cholesterol in sequence) were assessed in grade 3 obesity subjects undergoing BIBP (n = 10) and AGB (n = 10). Evaluations were performed before and 6 months after surgery. RESULTS: Subjects had similar values at baseline. Weight loss was similar in the two groups of subjects, and blood glucose, insulin levels, HOMA-IR, and triglycerides decreased in a similar way. In contrast, serum cholesterol, LDL cholesterol, non-HDL cholesterol, serum sitosterol, and campesterol levels decreased and lathosterol levels increased only in BIBP subjects, not in AGB subjects. A significant increase in 7-α-OH-cholesterol occurred only with BIBP; serum 27-OH-cholesterol decreased in both groups. CONCLUSIONS: Malabsorptive surgery specifically affects cholesterol levels, independent of weight loss and independent of glucose metabolism and insulin resistance. Decreased sterol absorption leads to decreased cholesterol and LDL cholesterol levels, accompanied by enhanced cholesterol synthesis and enhanced cholesterol catabolism. Compared with AGB, BIBP provides greater cholesterol lowering.

Long-term bone density evaluation in cerebrotendinous xanthomatosis: evidence of improvement after chenodeoxycholic acid treatment.

Cerebrotendinous xanthomatosis (CTX) is known to be associated with osteoporosis and a higher incidence of bone fractures. However, the underlying pathogenesis is still unknown, and the effects of long-term replacement therapy with chenodeoxycholic acid (CDCA) on bone mineral density (BMD) have not been fully investigated. We studied 11 CTX patients aged 13-43 years. We performed dual-energy X-ray absorptiometry and assessed serum cholestanol and 25-hydroxyvitamin D (25-OHD) concentrations both at the time of diagnosis and after long-term treatment with CDCA. At baseline, we found low BMD in nine patients, cholestanol elevation in all subjects, and 25-OHD decrease in nine. After a mean follow-up time of 30 months (range 24-36), no substantial clinical changes including bone fractures occurred; and we detected a significant increase of both planar and volumetric BMD as well as normalization of plasma cholestanol levels and increase of serum 25-OHD. Densitometric improvement following CDCA introduction was not correlated to changes of biochemical parameters. Our study confirms the presence of low bone mass in CTX and demonstrates that long-term CDCA treatment increases bone mineral content. In this respect, improvement of vitamin D intestinal absorption secondary to bile acid restoration could play an important role. Moreover, our data strongly suggest the utility of periodic bone density evaluation in CTX patients.

Effects of bile duct ligation and cholic acid treatment on fatty liver in two rat models of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease, one of the most prevalent liver disorders in Western countries, is characterized by hepatic accumulation of triglycerides. Bile acids have long been known to affect triglyceride homeostasis through a not completely understood mechanism. AIM: To analyse the effects of two different manipulations of bile acid circulation on non-alcoholic fatty liver disease. METHODS: Two animal models of non-alcoholic fatty liver disease were developed by either feeding rats with a choline deficient or with a high fat diet. After 4 weeks, rats were randomized to undergo either bile duct ligation, sham operation or cholic acid administration. RESULTS: During cholestasis there was an increased CYP7A1 expression, the rate limiting enzyme in bile acid synthesis, and a reduction of hepatic concentration of oxysterols, ligands of the liver X receptors. Target genes of the liver X receptors, involved in fatty acid and triglyceride synthesis, were down-regulated in association with decreased hepatic triglyceride content and improvement of fatty liver. Administration of cholic acid, ligand of farnesoid X receptor, also had a beneficial effect on fatty liver in rats on choline deficient diet. CONCLUSION: These results indicate that pharmacological approaches increasing the expression of CYP7A1 or stimulating farnesoid X receptor pathway could represent a promising treatment for non-alcoholic fatty liver disease.

Novel domain-selective ACE-inhibiting activity of synthetic growth hormone secretagogues.

The mechanisms of cardiovascular protective effects of ghrelin and its synthetic analogs are still largely unknown. Our first aim was to ascertain whether or not natural and synthetic ligands of GHS-R1a are capable of interfering with the activity of the renin-angiotensin system. Second, since polymorphisms in the ACE gene have been associated with Alzheimer's dementia (AD) and ACE is potentially involved in brain ß-amyloid degradation, we also investigated the state of ghrelin axis and inflammatory markers in patients with AD and vascular dementia (VaD). Desacyl ghrelin, hexarelin, EP80317, and GHRP-6 all significantly inhibited ACE activity in vitro; by comparison, the efficacies of ghrelin and MK-0677 were significantly lower, suggesting that ACE-inhibiting activity is unrelated to ligand affinity to GHS-R1a. ACE was capable of cleaving Aßin vitro, reducing its ability to aggregate in fibrillar Aß. Interestingly, this protective effect of ACE was blunted by enalapril but not hexarelin or EP80317. Desacyl ghrelin levels were lower in VaD subjects compared with AD and control subjects, whereas ghrelin and TNF-α levels were similar in all groups. VaD subjects demonstrated greater levels of mRNA for GHS-R1a, PPAR-γ and CD36 in peripheral blood lymphocytes compared with other groups. In conclusion, some GHSs are effective ACE-inhibitors, and this activity may contribute to their cardiovascular effects. Hexarelin or EP80317 do not inhibit the N-domain of ACE, which is also involved in the metabolism of ß-amyloid, suggesting the possibility of developing new antihypertensive drugs with improved therapeutic potential.

The use of stable and radioactive sterol tracers as a tool to investigate cholesterol degradation to bile acids in humans in vivo.

Alterations of cholesterol homeostasis represent important risk factors for atherosclerosis and cardiovascular disease. Different clinical-experimental approaches have been devised to study the metabolism of cholesterol and particularly the synthesis of bile acids, its main catabolic products. Most evidence in humans has derived from studies utilizing the administration of labeled sterols; these have several advantages over in vitro assay of enzyme activity and expression, requiring an invasive procedure such as a liver biopsy, or the determination of fecal sterols, which is cumbersome and not commonly available. Pioneering evidence with administration of radioactive sterol derivatives has allowed to characterize the alterations of cholesterol metabolism and degradation in different situations, including spontaneous disease conditions, aging, and drug treatment. Along with the classical isotope dilution methodology, other approaches were proposed, among which isotope release following radioactive substrate administration. More recently, stable isotope studies have allowed to overcome radioactivity exposure. Isotope enrichment studies during tracer infusion has allowed to characterize changes in the degradation of cholesterol via the "classical" and the "alternative" pathways of bile acid synthesis. Evidence brought by tracer studies in vivo, summarized here, provides an exceptional tool for the investigation of sterol metabolism, and integrate the studies in vitro on human tissue.