Pathogenetic mechanisms of amyloid A amyloidosis Simons, J. Paul; Al-Shawib, Raya; Ellmerich, Stephan ...
Proceedings of the National Academy of Sciences - PNAS,
10/2013, Letnik:
110, Številka:
40
Journal Article
Recenzirano
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Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils ...that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.
The hepatic predominating cytochrome P450, CYP3A4, plays an essential role in the detoxification of bile acids and is important in pathological conditions such as cholestasis where CYP3A4 is ...adaptively up-regulated. However, the mechanism that triggers the up-regulation of CYP3A4 is still not clear. In this study, using recombinant CYP3A4 and human liver microsomes, we demonstrate that CYP3A4 can metabolise lithocholic acid into 3-dehydrolithocholic acid, a potent activator of the nuclear receptors, pregnane X receptor and 1,25-dihydroxy vitamin D3 receptor, which are known to regulate the expression of CYP3A4. This process thus provides a feed-forward metabolism of toxic bile acid that may be of importance in maintaining bile acid homeostasis. We also provide evidence for a novel CYP3A4-mediated metabolic pathway of the secondary bile acid deoxycholic acid. Patients treated with the antiepileptic drug carbamazepine, a CYP3A4 inducer, had markedly elevated urinary excretion of 1β-hydroxydeoxycholic acid compared to healthy controls. The importance of CYP3A4 in this process was verified by incubations with recombinant CYP3A4 and human liver microsomes, both of which efficiently converted deoxycholic acid into 1β-hydroxydeoxycholic acid. Interestingly, CYP3A4 was also found to be active against the secondary bile acid ursodeoxycholic acid.
OBJECTIVESTo study the potential endogenous marker of CYP3A activity, 4β-hydroxycholesterol, and its relation to sex and the CYP3A5 geno/haplotypes and compare with CYP3A4/5 catalyzed 3-hydroxylation ...of quinine in the three major races.
METHODSThe plasma concentration of 4β-hydroxycholesterol was measured in healthy Tanzanians (n=138), Swedes (n=161) and Koreans (n=149) by gas chromatography–mass spectrometry. The metabolic ratio of quinine/3-hydroxyquinine in plasma 16-h post dose was determined by high performance liquid chromatography, previously reported in Tanzanians and Swedes, and now also in Koreans. The participants were genotyped for relevant alleles of CYP3A5.
RESULTSThe mean plasma concentrations of 4β-hydroxycholesterol in Koreans, Swedes and Tanzanians were 29.3, 26.8 and 21.9 ng/ml, respectively (P<0.01 between all three populations). Within all three populations there were significant differences in 4β-hydroxycholesterol levels between the CYP3A5 genotypes. Women had higher concentrations than men, but the difference was only significant in Tanzanians (P<0.001) and Koreans (P<0.00001). The quinine/3-hydroxyquinine metabolic ratio was significantly different in all three populations with the highest CYP3A activity in Koreans and the lowest in Tanzanians. Korean women had a lower metabolic ratio than men (P<0.00001). Significant correlations between 4β-hydroxycholesterol and quinine 3-hydroxylation were found in Tanzanians and Koreans.
CONCLUSIONClear differences in the activity of both CYP3A4 and CYP3A5 were shown in the three major human races. Both 4β-hydroxycholesterol and quinine/3-hydroxyquinine metabolic ratio showed a higher CYP3A activity in women than in men. The results give strong evidence that the plasma concentration of 4β-hydroxycholesterol may be used as an endogenous marker of CYP3A activity (CYP3A4+5).
Farnesoid X receptor knockout (Fxrsuperscript -/-) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxrsuperscript -/- mice ...differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxrsuperscript -/- mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxrsuperscript -/- mice, in particular hydroxylations of cholic acid in the 1{szligbeta}, 2{szligbeta}, 4{szligbeta}, 6alpha, 6{szligbeta}, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxrsuperscript -/- mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxrsuperscript -/- mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.
Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of ...additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier, and oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. Oxysterols are difficult to analyze by atmospheric pressure ionization mass spectrometry on account of the absence of basic or acidic functional groups in their structures. In this communication, we report a method for the derivatization and analysis of oxysterols by electrospray mass spectrometry. Oxysterols with a 3β-hydroxy-Δ
5 structure were converted by cholesterol oxidase to 3-oxo-Δ
4 steroids and then derivatized with the Girard P reagent to give Girard P hydrazones, which were subsequently analyzed by tandem mass spectrometry. The improvement in sensitivity for the analysis of 25-hydroxycholesterol upon oxidation and derivatization was over 1000.
Pregnenolone (PREG) and dehydroepiandrosterone (DHEA), and their respective sulfated forms PREGS and DHEAS, were among the first steroids to be identified in rodent brain. However, unreliable steroid ...isolation and solvolysis procedures resulted in errors, particularly in the case of brain steroid sulfates analyzed by radioimmunology or GC-MS of liberated free steroids. By using a solid-phase extraction recycling/elution procedure, allowing the strict separation of sulfated, free, and fatty acid esters of PREG and DHEA, PREGS and DHEAS, unlike free PREG, were not detected in rat and mouse brain and plasma. Conversely, considerable amounts of PREG and DHEA were released from unknown precursor(s) present in the lipoidal fraction, distinct from fatty acid ester conjugates. Chromatographic and mass spectrometric studies of the nature of the precursor(s) showed that autoxidation of brain cholesterol (CHOL) was responsible for the release of PREG and DHEA from the lipoidal fraction. When inappropriate protocols were used, CHOL was also the precursor of PREG and DHEA obtained from the fraction assumed to contain sulfated steroids. In contrast, free PREG was definitely confirmed as an endogenous steroid in rat brain. Our study shows that an early removal of CHOL from brain extracts coupled to well-validated extraction and fractionation procedures are prerequisites for reliable measurements of free and conjugated PREG and DHEA by GC-MS or other indirect methods.
Neutral steroids have traditionally been analyzed by gas chromatography/mass spectrometry (GC/MS) after necessary derivatization reactions. However, GC/MS is unsuitable for the analysis of many ...conjugated steroids and those with unsuspected functional groups. Here we describe an alternative analytical method specifically designed for the analysis of oxosteroids and those with a 3β-hydroxy-Δ5 or 5α-hydrogen-3β-hydroxy structure. Steroids were derivatized with Girard P (GP) hydrazine to give GP hydrazones, which are charged species and readily analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The resulting M+ ions were then subjected to high-energy collision-induced dissociation on a tandem time-of-flight instrument. The product ion spectra give structurally informative fragment ion patterns. The sensitivity of the analytical method is such that steroid structures can be determined from low-picogram (low-femtomole) amounts of sample. The utility of the method has been demonstrated by the analysis of oxysterols extracted from rat brain.
Rifampicin (rifampin) is a potent inducer of cytochrome P450 (CYP) 3A4. It was recently identified as a substrate of the polymorphic organic anion transporting polypeptide 1B1 (OATP1B1) expressed on ...the sinusoidal membrane of human hepatocytes. The present study aimed to investigate the possible association of single nucleotide polymorphisms (SNP) in the SLCO1B1 gene encoding for OATP1B1 with the inducing effect of rifampicin on hepatic CYP3A4. A total of 38 healthy volunteers who had participated in drug interaction studies with rifampicin were genotyped for the g.−11187G>A and c.521T>C SNPs in SLCO1B1, c.3435C>T SNP in ABCB1 and g.6986A>G SNP in CYP3A5. The plasma concentration of 4β-hydroxycholesterol, an endogenous marker of CYP3A4 activity, was measured before and after administration of 600 mg rifampicin once daily for 9–11 days. Treatment with rifampicin significantly increased the mean±SD plasma concentration of 4β-hydroxycholesterol from 55.2±17.9 ng/ml to 120.9±32.0 ng/ml (P<0.001). A large intersubject variability existed in the induction of CYP3A4 by rifampicin, but no associations were observed between the variability in induction and any of the polymorphisms studied. These data suggest that SLCO1B1 polymorphism does not affect the extent of induction of hepatic CYP3A4 by rifampicin, probably because other uptake transporters, such as OATP1B3, can compensate for reduced uptake of rifampicin by OATP1B1. However, the present study had sufficient power to detect only a considerably smaller rifampicin-induced increase in 4β-hydroxycholesterol in carriers of the SLCO1B1 c.521C allele compared to subjects with the reference genotype.
In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC)-mass ...spectrometry (MS) and tandem mass spectrometry (MS(n)) low levels of oxysterols (estimated 2-165 ng g(-1) wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7 alpha-, 7 beta-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 microg g(-1) in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng g(-1) in Ctx and 13 ng g(-1) in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng g(-1) in Ctx and 91 ng g(-1) in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg g(-1) in Ctx and 1.15 mg g(-1) in Sc) and its precursors were determined by gas chromatography-mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain development.
The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) ...and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).
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