Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases.

Copper transporting P-type (P1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD-1, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD-1, MBD-2, likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P1B-1-disorders and ongoing clinical trials.

Disparate Effects of Two Clerodane Diterpenes of Giant Goldenrod (Solidago gigantea Ait.) on Bacillus spizizenii.

New substances with antimicrobial properties are needed to successfully treat emerging human, animal, or plant pathogens. Seven clerodane diterpenes, previously isolated from giant goldenrod (Solidago gigantea) root, were tested against Gram-positive Bacillus subtilis, Bacillus spizizenii and Rhodococcus fascians by measuring minimal bactericidal concentration (MBC), minimal inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50). Two of them, Sg3a (a dialdehyde) and Sg6 (solidagoic acid B), were proved to be the most effective and were selected for further study. Bacillus spizizenii was incubated with the two diterpenes for shorter (1 h) or longer (5 h) periods and then subjected to genome-wide transcriptional analyses. Only a limited number of common genes (28 genes) were differentially regulated after each treatment, and these were mainly related to the restoration of cell membrane integrity and to membrane-related transports. Changes in gene activity indicated that, among other things, K+ and Na+ homeostasis, pH and membrane electron transport processes may have been affected. Activated export systems can be involved in the removal of harmful molecules from the bacterial cells. Inhibition of bacterial chemotaxis and flagellar assembly, as well as activation of genes for the biosynthesis of secondary metabolites, were observed as a general response. Depending on the diterpenes and the duration of the treatments, down-regulation of the protein synthesis-related, oxidative phosphorylation, signal transduction and transcription factor genes was found. In other cases, up-regulation of the genes of oxidation-reduction processes, sporulation and cell wall modification could be detected. Comparison of the effect of diterpenes with the changes induced by different environmental and nutritional conditions revealed several overlapping processes with stress responses. For example, the Sg6 treatment seems to have caused a starvation-like condition. In summary, there were both common and diterpene-specific changes in the transcriptome, and these changes were also dependent on the length of treatments. The results also indicated that Sg6 exerted its effect more slowly than Sg3a, but ultimately its effect was greater.

Effects of trientine and penicillamine on intestinal copper uptake: A mechanistic 64 Cu PET/CT study in healthy humans.

BACKGROUND AND AIMS: Trientine (TRI) and D-penicillamine (PEN) are used to treat copper overload in Wilson disease. Their main mode of action is thought to be through the facilitation of urinary copper excretion. In a recent study, TRI was noninferior to PEN despite lower 24-hour urinary copper excretion than PEN. We tested whether TRI and/or PEN also inhibit intestinal copper absorption. APPROACH AND RESULTS: Sixteen healthy volunteers were examined with positron emission tomography (PET)/CT 1 and 15 hours after an oral Copper-64 ( 64 Cu) dose. They then received 7 days of either PEN or TRI (trientine tetrahydrochloride), after which the 64 Cu PET/CT scans were repeated. Venous blood samples were also collected. Pretreatment to posttreatment changes of the hepatic 64 Cu uptake reflect the effect of drugs on intestinal absorption. 64 Cu activity was normalized to dose and body weight and expressed as the mean standard uptake value. TRI (n=8) reduced hepatic 64 Cu activity 1 hour after 64 Cu dose from 6.17 (4.73) to 1.47 (2.97) standard uptake value, p <0.02, and after 15 hours from 14.24 (3.09) to 6.19 (3.43), p <0.02, indicating strong inhibition of intestinal 64 Cu absorption. PEN (n=8) slightly reduced hepatic standard uptake value at 15 hours, from 16.30 (5.63) to 12.17 (1.44), p <0.04. CONCLUSIONS: In this mechanistic study, we show that TRI inhibits intestinal copper absorption, in addition to its cupriuretic effect. In contrast, PEN has modest effects on the intestinal copper absorption. This may explain why TRI and PEN are equally effective although urinary copper excretion is lower with TRI. The study questions whether the same therapeutic targets for 24-hour urinary excretion apply to both drugs.

Effects of tetrathiomolybdate on copper metabolism in healthy volunteers and in patients with Wilson disease.

BACKGROUND & AIMS: In Wilson disease (WD), copper accumulates in the liver and brain causing disease. Bis-choline tetrathiomolybdate (TTM) is a potent copper chelator that may be associated with a lower risk of inducing paradoxical neurological worsening than conventional therapy for neurologic WD. To better understand the mode of action of TTM, we investigated its effects on copper absorption and biliary excretion. METHODS: In a double-blind randomized setting, hepatic 64Cu activity was examined after orally administered 64Cu by PET/CT in 16 healthy volunteers before and after seven days of TTM treatment (15 mg/d) or placebo. Oral 64Cu was administered one hour after the final TTM dose. Changes in hepatic 64Cu activity reflected changes in intestinal 64Cu uptake. Additionally, in four patients with WD, the distribution of 64Cu in venous blood, liver, gallbladder, kidney, and brain was followed after i.v. 64Cu dosing for up to 68 hours before and after seven days of TTM (15 mg/day), using PET/MRI. Increased gallbladder 64Cu activity was taken as evidence of increased biliary 64Cu excretion. RESULTS: In healthy volunteers, TTM reduced intestinal 64Cu uptake by 82% 15 hours after the oral 64Cu dose. In patients with WD, gallbladder 64Cu activity was negligible before and after TTM, while TTM effectively retained 64Cu in the blood, significantly reduced hepatic 64Cu activity at all time-points and significantly reduced cerebral 64Cu activity two hours after the intravenous 64Cu dose. CONCLUSIONS: While we did not show an increase in biliary excretion of 64Cu following TTM administration, we demonstrated that TTM effectively inhibited most intestinal 64Cu uptake and retained 64Cu in the blood stream, limiting the exposure of organs like the liver and brain to 64Cu. IMPACT AND IMPLICATIONS: Bis-choline tetrathiomolybdate (TTM) is an investigational copper chelator being developed for the treatment of Wilson disease. In animal models of Wilson disease, TTM has been shown to facilitate biliary copper excretion. In the present human study, TTM surprisingly did not facilitate biliary copper excretion but instead reduced intestinal copper uptake to a clinically significant degree. Our study builds on our understanding of human copper metabolism and the mechanism of action of TTM.

Distribution of non-ceruloplasmin-bound copper after i.v. 64Cu injection studied with PET/CT in patients with Wilson disease.

Background & Aims: In Wilson disease (WD), copper accumulation and increased non-ceruloplasmin-bound copper in plasma lead to liver and brain pathology. To better understand the fate of non-ceruloplasmin-bound copper, we used PET/CT to examine the whole-body distribution of intravenously injected 64-copper (64Cu). Methods: Eight patients with WD, five heterozygotes, and nine healthy controls were examined by dynamic PET/CT for 90 min and static PET/CT up to 20 h after injection. We measured 64Cu activity in blood and tissue and quantified the kinetics by compartmental analysis. Results: Initially, a large fraction of injected 64Cu was distributed to extrahepatic tissues, especially skeletal muscle. Thus, across groups, extrahepatic tissues accounted for 45-58% of the injected dose (%ID) after 10 min, and 45-55% after 1 h. Kinetic analysis showed rapid exchange of 64Cu between blood and muscle as well as adipose tissue, with 64Cu retention in a secondary compartment, possibly mitochondria. This way, muscle and adipose tissue may protect the brain from spikes in non-ceruloplasmin-bound copper. Tiny amounts of cerebral 64Cu were detected (0.2%ID after 90 min and 0.3%ID after 6 h), suggesting tight control of cerebral copper in accordance with a cerebral clearance that is 2-3-fold lower than in muscle. Compared to controls, patients with WD accumulated more hepatic copper 6-20 h after injection, and also renal copper at 6 h. Conclusion: Non-ceruloplasmin-bound copper is initially distributed into a number of tissues before being redistributed slowly to the eliminating organ, the liver. Cerebral uptake of copper is extremely slow and likely highly regulated. Our findings provide new insights into the mechanisms of copper control. Impact and implications: Maintaining non-ceruloplasmin-bound copper within the normal range is an important treatment goal in WD as this "free" copper is considered toxic to the liver and brain. We found that intravenously injected non-ceruloplasmin-bound copper quickly distributed to a number of tissues, especially skeletal muscle, subcutaneous fat, and the liver, while uptake into the brain was slow. This study offers new insights into the mechanisms of copper control, which may encourage further research into potential new treatment targets. Clinical trial number: 2016-001975-59.

Antimicrobial Diterpenes from Rough Goldenrod (Solidago rugosa Mill.).

Solidago rugosa is one of the goldenrod species native to North America but has sporadically naturalized as an alien plant in Europe. The investigation of the root and leaf ethanol extracts of the plant using a bioassay-guided process with an anti-Bacillus assay resulted in the isolation of two antimicrobial components. Structure elucidation was performed based on high-resolution tandem mass spectrometric and one- and two-dimensional NMR spectroscopic analyses that revealed (-)-hardwickiic acid (Compound 1) and (-)-abietic acid (Compound 2). The isolates were evaluated for their antimicrobial properties against several plant pathogenic bacterial and fungal strains. Both compounds demonstrated an antibacterial effect, especially against Gram-positive bacterial strains (Bacillus spizizenii, Clavibacter michiganensis subsp. michiganensis, and Curtobacterium flaccumfaciens pv. flaccumfaciens) with half maximal inhibitory concentration (IC50) between 1 and 5.1 µg/mL (5-20 times higher than that of the positive control gentamicin). In the used concentrations, minimal bactericidal concentration (MBC) was reached only against the non-pathogen B. spizizenii. Besides their activity against Fusarium avenaceum, the highest antifungal activity was observed for Compound 1 against Bipolaris sorokiniana with an IC50 of 3.8 µg/mL.

Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders.

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in studying basic human and animal copper metabolism and copper-related disorders, such as Wilson disease (WD) and Menke's disease. A recent addition to this toolkit is 64-copper (64Cu) positron emission tomography (PET), combining the accurate anatomical imaging of modern computed tomography (CT) or magnetic resonance imaging (MRI) scanners with the biodistribution of the 64Cu PET tracer signal. This allows the in vivo tracking of copper fluxes and kinetics, thereby directly visualizing human and animal copper organ traffic and metabolism. Consequently, 64Cu PET is well-suited for evaluating clinical and preclinical treatment effects and has already demonstrated the ability to diagnose WD accurately. Furthermore, 64Cu PET/CT studies have proven valuable in other scientific areas like cancer and stroke research. The present article shows how to perform 64Cu PET/CT or PET/MR in humans. Procedures for 64Cu handling, patient preparation, and scanner setup are demonstrated here.

Clearance and production of ammonia quantified in humans by constant ammonia infusion - the effects of cirrhosis and ammonia-targeting treatments.

BACKGROUND & AIMS: Hyperammonaemia is a key pathological feature of liver disease and the primary driver of hepatic encephalopathy (HE). However, the relative roles of increased ammonia production and reduced clearance are poorly understood as is the action of ammonia-targeting drugs for HE. We aimed to quantify whole-body ammonia metabolism in healthy persons and patients with cirrhosis and to validate our method by examining the effects of glycerol phenylbutyrate and lactulose + rifaximin treatment. METHODS: Ten healthy men and ten male patients with cirrhosis were investigated by 90-minute constant ammonia infusion to achieve steady-state plasma ammonia. Whole-body ammonia clearance was calculated as infusion rate divided by steady-state concentration increase and ammonia production was calculated as clearance multiplied by baseline ammonia concentration. Participants were re-investigated after the ammonia-targeting interventions. RESULTS: In healthy persons, ammonia clearance was 3.5 (3.1-3.9) L/min and ammonia production was 49 (35-63) µmol/min. Phenylbutyrate increased clearance by 11% (4-19%, p = 0.009). In patients with cirrhosis, ammonia clearance was 20% lower at 2.7 (2.1-3.3) L/min (p = 0.02) and production was nearly threefold higher at 131 (102-159) µmol/min (p <0.0001). Lactulose + rifaximin reduced production by 20% (2-37%, p = 0.03). The infusion was generally well-tolerated apart from in one hyperammonaemic patient, with cirrhosis and possible bleeding unrelated to the infusion, who developed clinical HE that reverted when infusion was discontinued. CONCLUSIONS: Whole-body ammonia clearance and production may be measured separately using the described technique. This technique identified a lower clearance and a higher production of ammonia in patients with cirrhosis, and showed that phenylbutyrate increases clearance, whereas lactulose + rifaximin reduces production. IMPACT AND IMPLICATIONS: High blood ammonia plays a key role in cirrhosis-related brain dysfunction. However, the relative roles of reduced ammonia clearance and increased ammonia production are poorly understood as is the action of ammonia-targeting treatments. This study presents a relatively simple test to measure ammonia metabolism. By using this test, it was possible to show that patients with cirrhosis exhibit decreased ammonia clearance and increased ammonia production compared to healthy persons, and to quantify the unique effects of different ammonia-targeting treatments. The test described herein may be used to examine a range of questions related to normal physiology, pathophysiology and the mechanisms of action of ammonia-targeting treatments. CLINICAL TRIAL NUMBER: ClinicalTrials.gov (1-16-02-297-20).

The association between soluble CD163, disease severity, and ursodiol treatment in patients with primary biliary cholangitis.

INTRODUCTION: The macrophage activation marker soluble (s)CD163 is associated with disease severity and prognosis in patients with primary biliary cholangitis (PBC). Ursodeoxycholic acid (UDCA) treatment attenuates fibrosis progression in PBC patients, but its effect on macrophage activation is unclear. We examined the effect of UDCA on macrophage activation, as determined by sCD163 levels. METHODS: We included 2 cohorts of PBC patients; 1 cohort with prevalent PBC patients, and 1 cohort of incident PBC patients before start of UDCA treatment and with follow-up after 4 weeks and 6 months. We measured sCD163 and liver stiffness in both cohorts. Further, we measured sCD163 and TNF-α shedding in vitro in monocyte-derived macrophages after UDCA and lipopolysaccharide incubation. RESULTS: We included 100 patients with prevalent PBC [93% women, median age 63 y (interquartile range: 51-70)] and 47 patients with incident PBC [77% women, median age 60 y (49-67)]. Prevalent PBC patients had a lower median sCD163 of 3.54 mg/L (2.77-4.72) than incident PBC patients with a median sCD163 of 4.33 mg/L (2.83-5.99) at inclusion. Patients with an incomplete response to UDCA and patients with cirrhosis had higher sCD163 than responders to UDCA and noncirrhosis patients. After 4 weeks and 6 months of UDCA treatment median sCD163 decreased by 4.6% and 9.0%, respectively. In in vitro experiments, UDCA attenuated shedding of TNF-α, but not sCD163, from monocyte-derived macrophages. CONCLUSION: In PBC patients, sCD163 levels correlated with liver disease severity and treatment response to UDCA. Further, after 6 months of UDCA treatment, we observed a decrease in sCD163, which may be related to the treatment.

Nine-dimensional bioprofiles of Tunisian sages (Salvia officinalis, S. aegyptiaca and S. verbenaca) by high-performance thin-layer chromatography - effect-directed analyses.

Ethyl acetate extracts of Tunisian Salvia aegyptiaca and S. verbenaca aerial parts and S. officinalis leaves were examined via bioanalytical profiling using high-performance thin-layer chromatography (HPTLC) combined with nine bioactivity assays, namely antibacterial (Aliivibrio fischeri, Bacillus subtilis, and Rhodococcus fascians), antifungal (Bipolaris sorokiniana, and Fusarium avenaceum), radical scavenging (DPPH•), and enzyme inhibitory (α-glucosidase, acetylcholinesterase, and lipase) ones. The screening, using toluene - ethyl acetate - methanol 6:3:0.5 (V/V/V) as a mobile phase, revealed five bioactive zones (a-e) that were analyzed by HPTLC-electrospray ionization-mass spectrometry (ESI-MS). Zones b and c, observed exclusively in S. officinalis, were active in all assays except α-glucosidase, and only c inhibited F. avenaceum. Compounds in these zones were identified by HPLC-high resolution tandem MS (LC-HRMS/MS) as rosmanol/epi-rosmanol and methyl carnosate, respectively. In the bioactive zones a and e, corosolic/maslinic acid and ursolic/oleanolic acid isomer pairs were present, which could be identified in all three Salvia species after their HPTLC separation using pre-chromatographic derivatization with iodine and MS detection. The triterpenes inhibited B. subtilis and R. fascians bacteria and α-glucosidase enzyme. Linoleic and linolenic acids were detected in zone d, which showed strong lipase inhibition in all three sage species.

Outcome of elexacaftor/tezacaftor/ivacaftor therapy in patients with cystic fibrosis and solid organ transplantation.

We report a case series of four patients with cystic fibrosis (CF) and previous solid organ transplantation (SOT) receiving elexacaftor/tezacaftor/ivacaftor therapy for 6 months or more. Data was collected retrospectively. The treatment was well tolerated and all patients reported subjective improvements.

Trientine tetrahydrochloride versus penicillamine for maintenance therapy in Wilson disease (CHELATE): a randomised, open-label, non-inferiority, phase 3 trial.

BACKGROUND: Wilson disease is an inherited disorder of copper transport. Whereas penicillamine is used therapeutically to re-establish copper balance, trientine is indicated for patients with penicillamine intolerance. We aimed to compare penicillamine with trientine tetrahydrochloride (TETA4) for maintenance therapy in patients with Wilson disease. METHODS: We conducted a randomised, open-label, non-inferiority, phase 3 trial at 15 health-care centres across nine countries (patients were recruited from 13 of these health-care centres across Brazil, Europe, and the USA). We enrolled patients aged 18-75 years with stable Wilson disease who were treated for at least 1 year with penicillamine. Patients entered a 12-week period to determine stability through clinical assessment by site investigators and predefined thresholds for serum non-caeruloplasmin-bound copper (NCC; by an exchangeable copper assay; 25-150 µg/L), 24 h urinary copper excretion (100-900 µg/24 h), and alanine aminotransferase (ALT; <2 × upper limit of normal). Stable patients were randomly assigned (1:1) to continue receiving the maintenance twice daily dose of oral penicillamine or switched mg-for-mg to oral TETA4 centrally with a web-based system using minimisation. The primary endpoint, assessed 24 weeks after randomisation, was NCC by speciation assay. The non-inferiority margin of mean difference in NCC by speciation assay was -50 µg/L, as estimated by a general linear model for repeated visits, adjusted for baseline values. Further data on safety and efficacy were collected during a 24-week extension period. Data were analysed using an intention-to-treat approach. Safety was assessed in all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, NCT03539952 (active, not recruiting). FINDINGS: Between June 4, 2018, and March 10, 2020, 77 patients were screened. 53 patients were randomly assigned (27 to the penicillamine group and 26 to the TETA4 group). After 24 weeks, the mean difference in serum NCC by speciation assay between the penicillamine group and TETA4 group was -9·1 µg/L (95% CI -24·2 to 6·1), with the lower limit of the 95% CI within the defined non-inferiority margin. At 24 weeks, urinary copper excretion was lower with TETA4 than with penicillamine (mean difference 237·5 µg/24 h (99% CI 115·6 to 359·4). At 48 weeks, TETA4 remained non-inferior to penicillamine in terms of NCC by speciation assay (mean difference NCC -15·5 µg/L [95% CI -34·5 to 3·6]). Urinary copper excretion at 48 weeks remained in the expected range for well treated patients in both study groups, and the mean difference (124·8 µg/24 h [99% CI -37·6 to 287·1]) was not significantly different. At 24 weeks and 48 weeks, masked clinical adjudication of stability assessed by three independent clinicians confirmed clinical stability (100%) of all participants, in agreement with the stability seen with the NCC by speciation assay. There were no notable changes in either the Clinical Global Impression of Change or Unified Wilson Disease Rating Scale (neurological assessment) from baseline (pre-randomisation) at weeks 24 and 48. The mean change in serum total copper from baseline to 24 weeks was 17·6 µg/L (99% CI -9·5 to 44·7) with penicillamine and -6·3 µg/L (-34·7 to 22·1) with TETA4, and the mean change in serum total caeruloplasmin from baseline to 24 weeks was 1·8 mg/L (-19·2 to 22·8) with penicillamine and -2·2 mg/L (-6·1 to 1·7) with TETA4. All liver enzymes were similar at 24 weeks and 48 weeks, with the exception of elevated ALT concentration at 48 weeks for patients in the TETA4 group. Penicillamine was associated with three post-randomisation serious adverse events (leukopenia, cholangiocarcinoma, and hepatocellular cancer); none were reported for TETA4. The most common treatment-emergent adverse events were headache for penicillamine (five [19%] of 27 patients vs two [8%] of 26) and abdominal pain for TETA4 (one [4%] vs four [15%]); all treatment-emergent adverse events resolved and were mild to moderate. One patient developed a rash with TETA4 that resolved on discontinuation of therapy. INTERPRETATION: The efficacy of TETA4 as oral maintenance therapy was non-inferior to penicillamine and well tolerated in adults with Wilson disease. FUNDING: Orphalan.

Case report: Huppke-Brendel syndrome in an adult, mistaken for and treated as Wilson disease for 25 years.

Background: Huppke-Brendel (HB) syndrome is an autosomal recessive disease caused by variants in the SLC33A1 gene. Since 2012, less than ten patients have been reported, none survived year six. With neurologic involvement and ceruloplasmin deficiency, it may mimic Wilson disease (WD). Objectives and methods: We report the first adult patient with HB. Results: The patient suffered from moderate intellectual disability, partial hearing loss, spastic ataxia, hypotonia, and unilateral tremor of parkinsonian type. At age 29, she was diagnosed with WD based on neurology, elevated 24H urinary copper, low ceruloplasmin, and pathological 65Cu test. Approximately 25 years later, genetic testing did not support WD or aceruloplasminemia. Full genome sequencing revealed two likely pathogenic variants in SLC33A1 which combined with re-evaluation of neurologic symptoms and MRI suggested the diagnosis of HB. Conclusion: Adult patients with HB exist and may be confused with WD. Low ceruloplasmin and the absence of ATP7B variants should raise suspicion.

Effect of oral zinc regimens on human hepatic copper content: a randomized intervention study.

Zinc inhibits intestinal copper uptake, an effect utilized for treating Wilson's disease (WD). We used copper-64 (64Cu) PET/CT to examine how much four weeks of treatment with different zinc regimens reduced the hepatic 64Cu content after oral 64Cu administration and test if alternative regimens were noninferior to the standard regimen of zinc acetate 50 mg × 3 daily. Forty healthy persons were randomized to four different zinc protocols. The WD standard treatment zinc acetate 50 mg × 3 reduced the hepatic 64Cu content from 26.9 ± 7.5% to 13.3 ± 5.6% of the administered 64Cu. Zinc gluconate 50 mg × 3 was noninferior (P = 0.02) (35.8 ± 9.0% to 17.4 ± 7.5%). Zinc acetate 150 mg × 1 (33.1 ± 9.9% to 17.4 ± 7.5%) and zinc gluconate 150 mg × 1 (28.1 ± 6.7% to 22.0 ± 6.7%) were less effective. These effects were intra- and inter-individually highly variable, and 14% had no effect of any zinc regimen, which may explain disparities in zinc treatment efficacy in WD patients.

High-performance thin-layer chromatography - antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.).

The present work introduces a high-performance thin-layer chromatography (HPTLC)-direct bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLC-EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC-mass spectrometry (MS), and bioassay-guided column chromatographic (preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform - ethyl acetate - methanol 15:3:2, V/V/V) was changed (to n-hexane - isopropyl acetate - methanol - acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1-2 and 3-4). Compounds 1 and 3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R. fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC50) values in the range of 32.3-64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.

Low cerebral energy metabolism in hepatic encephalopathy reflects low neuronal energy demand. Role of ammonia-induced increased GABAergic tone.

Hepatic encephalopathy (HE) is a frequent and devastating but generally reversible neuropsychiatric complication secondary to chronic and acute liver failure. During HE, brain energy metabolism is markedly reduced and it remains unclear whether this is due to external or internal energy supply limitations, or secondary to depressed neuronal cellular functions - and if so, which mechanisms that are in play. The extent of deteriorated cerebral function correlates to blood ammonia levels but the metabolic link to ammonia is not clear. Early studies suggested that high levels of ammonia inhibited key tricarboxylic acid (TCA) cycle enzymes thus limiting mitochondrial energy production and oxygen consumption; however, later studies by us and others showed that this is not the case in vivo. Here, based on a series of translational studies from our group, we advocate the view that the low cerebral energy metabolism of HE is likely to be caused by neuronal metabolic depression due to an elevated GABAergic tone rather than by restricted energy availability. The increased GABAergic tone seems to be secondary to synthesis of large amounts of glutamine in astrocytes for detoxification of ammonia with the glutamine acting as a precursor for elevated neuronal synthesis of vesicular GABA.

Separation and detection of apricot leaf triterpenes by high-performance thin-layer chromatography combined with direct bioautography and mass spectrometry.

Prunus armeniaca leaf extract was screened for antibacterial compounds by high-performance thin-layer chromatography (HPTLC)-direct bioautography using a Gram-positive Bacillus subtilis bacterium. Six chromatographic zones exhibited characteristic bioactivity. Five of them also appeared after derivatization with vanillin-sulfuric acid reagent and could be characterized with HPTLC-electrospray ionization (ESI)-mass spectrometry (MS), suggesting the presence of triterpenoids and the fatty acids linolenic and palmitic acid. To confirm the identification of triterpenoids an HPTLC method using in situ pre-chromatographic derivatization with iodine was developed to separate the closely related triterpenoids. After development, the iodine could be eliminated from the chromatogram (verified by HPTLC-MS), making it suitable for the B. subtilis assay. Ursolic acid, oleanolic acid, betulinic acid, corosolic acid, and maslinic acid were discovered for the first time as antibacterial components of P. armeniaca leaves. Their presence was proved also by 2D-HPTLC combined with intermediate in situ derivatization by iodine.

The galactose elimination capacity test to monitor liver disease course in patients with Wilson's disease.

BACKGROUND: The prognosis of Wilson's disease (WD) has changed radically since the introduction of medical therapy with chelators and zinc. However, there is an unmet need for methods to evaluate the long-term treatment response and the liver disease progression in order to identify treatment failures. The galactose elimination capacity test (GEC) is a physiological measure of the total metabolic capacity of the liver, and a strong predictor of long- and short-term mortality in patients with liver cirrhosis. Our aim was to investigate if the GEC test is useful for evaluation of treatment response and prediction of treatment failures in WD patients. METHODS: We included all patients with WD in Denmark from 1992 through 2017 and retrieved data on GEC along with data on transplantation and death. RESULTS: In total, 37 patients had completed one or more GEC tests. Of these, 31 were alive (three transplanted) and six were dead (two transplanted). A total of 24 patients had completed more than one GEC test. All 18 alive, nontransplanted patients showed improvement in GEC values following onset of treatment, except one patient, who was clinically confirmed with treatment failure. All six patients who underwent liver transplantation or died had a prior decline in their GEC. The difference in GEC development between patients alive and not transplanted and patients dead or transplanted was significant (p < .001). Index GEC values could not predict transplantation or death (p = .26). CONCLUSION: The GEC test is a promising tool for monitoring treatment response and identifying treatment failures in patients with WD.

The pathophysiology of Wilson's disease visualized: A human 64 Cu PET study.

BACKGROUND AND AIMS: Wilson's disease (WD) is a genetic disease with systemic accumulation of copper that leads to symptoms from the liver and brain. However, the underlying defects in copper transport kinetics are only partly understood. We sought to quantify hepatic copper turnover in patients with WD compared with heterozygote and control subjects using PET with copper-64 (64 Cu) as a tracer. Furthermore, we assessed the diagnostic potential of the method. APPROACH AND RESULTS: Nine patients with WD, 5 healthy heterozygote subjects, and 8 healthy controls were injected with an i.v. bolus of 64 Cu followed by a 90-min dynamic PET scan of the liver and static whole-body PET/CT scans after 1.5, 6, and 20 h. Blood 64 Cu concentrations were measured in parallel. Hepatic copper retention and redistribution were evaluated by standardized uptake values (SUVs). At 90 min, hepatic SUVs were similar in the three groups. In contrast, at 20 h postinjection, the SUV in WD patients (mean ± SEM, 31 ± 4) was higher than in heterozygotes (24 ± 3) and controls (21 ± 4; p < 0.001). An SUV-ratio of hepatic 64 Cu concentration at 20 and 1.5 h completely discriminated between WD patients and control groups (p < 0.0001; ANOVA). By Patlak analysis of the initial 90 min of the PET scan, the steady-state hepatic clearance of 64 Cu was estimated to be slightly lower in patients with WD than in controls (p = 0.04). CONCLUSIONS: 64 Cu PET imaging enables visualization and quantification of the hepatic copper retention characteristic for WD patients. This method represents a valuable tool for future studies of WD pathophysiology, and may assist the development of therapies, and accurate diagnosis.