Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease.

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.

Down-Regulation of Collagen Hydroxylation in Colorectal Liver Metastasis.

Collagen is significantly upregulated in colorectal liver metastasis (CRLM) compared to liver tissue. Expression levels of specific collagen types in CRLM resemble those in colorectal cancer (CRC) and colon tissue. We investigated whether the collagen hydroxylation pattern from the primary tumor also migrates with the metastatic tumor. The degree of collagen alpha-1(I) hydroxylation in colon, CRC, liver, and CRLM tissue of the same individuals (n = 14) was studied with mass spectrometry. The degree of hydroxylation was investigated in 36 collagen alpha-1(I) peptides, covering 54% of the triple helical region. The degree of hydroxylation in liver tissue was similar to that in colon tissue. The overall degree of hydroxylation was significantly lower (9 ± 14%) in CRC tissue and also significantly lower (12 ± 22%) in CRLM tissue compared to colon. Furthermore, eleven peptides with a specific number of hydroxylations are significantly different between CRLM and liver tissue; these peptides could be candidates for the detection of CRLM. For one of these eleven peptides, a matching naturally occurring peptide in urine has been identified as being significantly different between patients suffering from CRLM and healthy controls. The hydroxylation pattern in CRLM resembles partly the pattern in liver, primary colorectal cancer and colon.

Identification of a Collagen Marker in Urine Improves the Detection of Colorectal Liver Metastases.

Previously, we reported a combination of an urine collagen alpha-1(I) natural occurring peptide (NOP) AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP (AGP) and serum carcinoembryonic antigen (CEA) to have the potential to detect colorectal liver metastasis (CRLM). The combined method requires further adaption for better sensitivity and specificity prior to clinical implementation. This mass spectrometry study aimed to identify additional collagen NOPs in urine and determine the most discriminating NOP panel. We improved the combined method on the basis of analysis of urine samples from 100 healthy controls and 100 CRLM patients. Two additional NOPs were identified: GPPGEAGK(-OH)P(-OH)GEQGVP(-OH)GDLGAP(-OH)GP (GPP), collagen alpha-1(I), and GNDGARGSDGQPGPP(-OH)GP(-OH)P(-OH)GTAGFP(-OH)GSP(-OH)GAK(-OH)GEVGP (GND), collagen alpha-1(III). A molecular model combining NOPs (AGP, GPP, and GND) and CEA was generated. Molecules that did not contribute significantly were removed, resulting in a model consisting of GND and CEA. With this model, 88% sensitivity and 88% specificity were reached in the discovery set and 75% sensitivity and 100% specificity in the validation set (control, n = 12; CRLM, n = 10). The AUC of the ROC curve is significantly higher than the current model based on AGP and CEA (p = 3.3 × 10-4). The new model performs better than the currently used techniques in the clinic that have a 57-70% sensitivity and a 90-96% specificity.

Collagen analysis with mass spectrometry.

Mass spectrometry-based techniques can be applied to investigate collagen with respect to identification, quantification, supramolecular organization, and various post-translational modifications. The continuous interest in collagen research has led to a shift from techniques to analyze the physical characteristics of collagen to methods to study collagen abundance and modifications. In this review, we illustrate the potential of mass spectrometry for in-depth analyses of collagen.

One electron less or one proton more: how do they differ?

From the NIST website and the literature, we have collected the Ionisation Energies (IE) of 3,052 and the Proton Affinities (PA) of 1,670 compounds. For 614 of these, both the IE and PA are known; this enables a study of the relationships between these quantities for a wide variety of molecules. From the IE and PA values, the hydrogen atom affinities (HA) of molecular ions M•+ may also be assessed. The PA may be equated to the heterolytic bond energy of [MH]+ and HA to the homolytic bond energy. Plots of PA versus IE for these substances show (in agreement with earlier studies) that, for many families of molecules, the slope of the ensuing line is less negative than -1, i.e. changes in the PA are significantly less than the concomitant opposite changes in IE. At one extreme (high PA, low IE) are the metals, their oxides and hydroxides, which show a slope of close to -1, at the other extreme (low PA, high IE) are the hydrogen halides, methyl halides and noble gases, which show a slope of ca. -0.3; other molecular categories show intermediate behaviour. One consequence of a slope less negative than -1 is that the changes in ionic enthalpies of the protonated species more closely follow the changes in the enthalpies of the neutral molecules compared with changes in the ion enthalpies of the corresponding radical cations. This is consistent with findings from ab initio calculations from the literature that the incoming proton, once attached to the molecule, may retain a significant amount of its charge. These collected data allow a comparison of the thermodynamic stability of protonated molecules in terms of their homolytic or heterolytic bond cleavages. Protonated nitriles are particularly stable by virtue of the very large hydrogen atom affinities of their radical cations.

Identification of 4-Hydroxyproline at the Xaa Position in Collagen by Mass Spectrometry.

Collagen has a triple helix form, structured by a [-Gly-Xaa-Yaa-] repetition, where Xaa and Yaa are amino acids. This repeating unit can be post-translationally modified by enzymes, where proline is often hydroxylated into hydroxyproline (Hyp). Two Hyp isomers occur in collagen: 4-hydroxyproline (4Hyp, Gly-Xaa-Pro, substrate for 4-prolyl hydroxylase) and 3-hydroxyproline (3Hyp, Gly-Pro-4Hyp, substrate for 3-prolyl hydroxylase). If 4Hyp is lacking at the Yaa position, then Pro at the Xaa position should remain unmodified. Nevertheless, in literature 41 positions have been described where Hyp occurs at the Xaa position (?xHyp) lacking an adjacent 4Hyp. We report four additional positions in liver and colorectal liver metastasis tissue (CRLM). We studied the sequence commonalities between the 45 known positions of ?xHyp. Alanine and glutamine were frequently present adjacent to ?xHyp. We showed that proline, position 584 in COL1A2, had a lower rate of modification in CRLM than in healthy liver. The isomeric identity of ?xHyp, that is, 3- and/or 4Hyp, remains unknown. We present a proof of principle identification of ?xHyp. This identification is based on liquid chromatography retention time differences and mass spectrometry using ETD-HCD fragmentation, complemented by ab initio calculations. Both techniques identify ?xHyp at position 584 in COL1A2 as 4-hydroxyproline (4xHyp).

Up-regulation of collagen proteins in colorectal liver metastasis compared with normal liver tissue.

Changes to extracellular matrix (ECM) structures are linked to tumor cell proliferation and metastasis. We previously reported that naturally occurring peptides of collagen type I are elevated in urine of patients with colorectal liver metastasis (CRLM). In the present study, we took an MS-based proteomic approach to identify specific collagen types that are up-regulated in CRLM tissues compared with healthy, adjacent liver tissues from the same patients. We found that 19 of 22 collagen-α chains are significantly up-regulated (p < 0.05) in CRLM tissues compared with the healthy tissues. At least four collagen-α chains were absent or had low expression in healthy colon and adjacent tissues, but were highly abundant in both colorectal cancer (CRC) and CRLM tissues. This expression pattern was also observed for six noncollagen colon-specific proteins, two of which (CDH17 and PPP1R1B/DARP-32) had not previously been linked to CRLM. Furthermore, we observed CRLM-associated up-regulation of 16 proteins (of 20 associated proteins identified) known to be required for collagen synthesis, indicating increased collagen production in CRLM. Immunohistochemistry validated that collagen type XII is significantly up-regulated in CRLM. The results of this study indicate that most collagen isoforms are up-regulated in CRLM compared with healthy tissues, most likely as a result of an increased collagen production in the metastatic cells. Our findings provide further insight into morphological changes in the ECM in CRLM and help explain the finding of tumor metastasis-associated proteins and peptides in urine, suggesting their utility as metastasis biomarkers.

Proton affinities and ion enthalpies.

Proton affinities of a number of alkyl acetates (CH3-C(=O)-OR) and of methyl alkanoates (R-C(=O)-OCH3, R=H, alkyl) have been assembled from the literature or measured using the kinetic method. It was observed that the proton affinities for the isomeric species CH3-C(=O)-OR and R-C(=O)-OCH3 are almost identical, an unexpected result as the charge in these protonated ester molecules is largely at the keto carbon atom and so this site should be more sensitive to alkyl substitution. Analysis of the data, including those from lone pair ionisation and core-electron ionisation experiments available from the literature, indicate that after protonation, extensive charge relaxation (or polarisation) takes place (as is also the case, according to the literature, after core-electron ionisation). By contrast, after lone pair ionisation, which results in radical cations, such relaxation processes are relatively less extensive. As a consequence, changes in ion enthalpies of these protonated molecules follow more closely the changes in neutral enthalpies, compared with changes in enthalpies of the corresponding radical cations, formed by electron detachment. Preliminary analyses of published energetic data indicate that the above finding for organic esters may well be another example of a more general phenomenon.

Decellularization of Whole Human Liver Grafts Using Controlled Perfusion for Transplantable Organ Bioscaffolds.

Liver transplantation is the only effective treatment for end-stage liver disease, but absolute donor shortage remains a limiting factor. Recent advances in tissue engineering focus on generation of native extracellular matrix (ECM) by decellularized complete livers in animal models. Although proof of concept has been reported for human livers, this study aims to perform whole liver decellularization in a clinically relevant series using controlled machine perfusion. In this study, we describe a mild nondestructive decellularization protocol, effective in 11 discarded human whole liver grafts to generate constructs that reliably maintain hepatic architecture and ECM components using machine perfusion, while completely removing cellular DNA and RNA. The decellularization process preserved the ultrastructural ECM components confirmed by histology, electron microscopy, and proteomic analysis. Anatomical characteristics of the native microvascular network and biliary drainage of the liver were confirmed by contrast computed tomography scanning. Decellularized vascular matrix remained suitable for normal suturing and no major histocompatibility complex molecules were detected, suggesting absence of allo-reactivity when used for transplantation. After extensive washing, decellularized scaffolds were nontoxic for cells after reseeding human mesenchymal stromal or umbilical vein endothelial endothelium cells. Indeed, evidence of effective recellularization of the vascular lining was obtained. In conclusion, we established an effective method to generate clinically applicable liver scaffolds from human discarded whole liver grafts and show proof of concept that reseeding of normal human cells in the scaffold is feasible. This supports new opportunities for bioengineering of transplantable grafts in the future.

Interaction of metal cations with functionalised hydrocarbons in the gas phase: further experimental evidence for solvation of metal ions by the hydrocarbon chain.

Relative affinity measurements of monovalent metal ions (= Li(+), Cu(+) and Ag(+)) towards aliphatic amines, alcohols and methyl alkanoates (P) have been performed using the kinetic method on the dissociation of metal bound dimer ions of the type P(1)-M(+)-P(2). It was found that the cations' affinity towards long chain (≥C(4) chain length) n- and s-alkylamines, n-alkanols and methyl n- alkanoates was unexpectedly enhanced. This is attributed to a bidentate interaction of the metal ion with the amine, alcohol or ester functional group and the aliphatic chain, paralleling earlier observations on metal bound nitriles. Methyl substitution at the functional group (s-alkylamines compared with n-alkylamines) serves to strengthen only the N•••M(+) bond, and this can be rationalized by the larger proton affinities of s-alkylamines compared to n-alkylamines. This substitution, however, has no effect on the metal ion-hydrocarbon bond. In contrast, methyl substitution remote from the functional group, as in iso-pentylamine, does lead to strengthening of the metal ion-hydrocarbon bond. The cuprous ion affinity of hexadecylamine, C(16)H(33)NH(2) was found to be as large as that for ethylenediamine (352 kJ mol(-1)), known to be a strong copper binding agent. It is argued that such a metal ion-hydrocarbon interaction does not occur in the metal bound dimers.

Hydroxylated collagen peptide in urine as biomarker for detecting colorectal liver metastases.

The clinical efficacy of carcinoembryonic antigen (CEA) as a marker of colorectal liver metastasis is limited, motivating a search for new biomarkers. Recently, urine proteomic analysis revealed AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP (AGP), a promising peptide for this application. This study aimed to determine whether combining urine AGP testing with serum CEA analyses improves the sensitivity of detecting colorectal liver metastases. Urine samples from 100 patients with CRLM were collected prospectively and compared to three control groups: healthy kidney donors, patients who were relapse-free for 24 months after curative CRLM surgery, and primary colorectal cancer patients. A stable isotope labeled peptide standard was used to quantify the abundance of AGP in urine samples by selective reaction monitoring. Combined testing of urine AGP levels and serum CEA levels revealed a significantly increased sensitivity compared to CEA alone (85% vs. 68%, P<0.001; specificity 84% and 91%, respectively). No correlation was found between CEA and AGP-positive test results within individual patients (r(2) = 0.08). Urine AGP testing was negative in the three control groups. These results indicate that collagen-derived urine AGP peptide with a specific hydroxylation pattern combined with serum CEA levels may significantly improve the detection of colorectal liver metastases in patients at risk.

Interaction of metal cations with alkylnitriles in the gas phase: solvation of metal ions by the hydrocarbon chain.

Relative affinity measurements of monovalent metal ions (M = Li(+), Na(+), Cu(+)and Ag(+)) toward aliphatic nitriles have been performed using the kinetic method by dissociation of metal bound dimer ions of the type R1C≡N-M(+)-N≡CR(2). It is found, particularly for Cu(+) and Ag(+), that the affinity towards nitriles having long chains (>C(6)) is markedly enhanced. This is attributed to a bidentate interaction of the metal ion with the nitrile moiety and the aliphatic chain. Theoretical calculations on the copper complexes show that these bidentate structures enjoy about 30% greater copper ion affinities compared to their linear counterparts. Such aliphatic interactions also play a major role in the dissociation chemistry of copper bound tetramers of the kind (RC≡N)(4)Cu(2+∙) where the long aliphatic chain R curls around the copper ion to facilitate electron transfer or a redox reaction to produce (RC≡N)(2)Cu(+) + RC≡N(+∙) + RC≡N.

Collagen peptides in urine: a new promising biomarker for the detection of colorectal liver metastases.

INTRODUCTION: For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls. MATERIALS AND METHODS: Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM's) were developed on a UPLC-MS/MS system. RESULTS: Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%. CONCLUSION: Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.

Dried matrix on paper disks: the next generation DBS microsampling technique for managing the hematocrit effect in DBS analysis.

BACKGROUND: The hematocrit effect is a hurdle for successful introduction of the dried blood spot (DBS) in a regulated environment. Recently, attempts were taken to overcome the hematocrit effect by whole-cut DBS analysis. This paper presents the next-generation whole-cut DBS; dried matrix on paper disks (DMPD). RESULTS: DMPD eliminated the hematocrit effect and demonstrated better accuracy and precision than regular DBS with partial punching. Observed accuracy and precision were 6.0 and 2.3% for DMPD, respectively, and -10.4 and 17.1%, for DBS, respectively. CONCLUSION: The DMPD technique performed better than regular DBS by eliminating the hematocrit effect related blood volume bias. Although this effect was not observed with DMPD, a systematic error of 6.0% was detected and further technical development of DMPD could improve the performance.

Analytical investigations of toxic p-phenylenediamine (PPD) levels in clinical urine samples with special focus on MALDI-MS/MS.

Para-phenylenediamine (PPD) is a common chromophoric ingredient in oxidative hair-dyes. In some African countries like Sudan, Egypt and Morocco but also in India this chemical is used alone or in combination with colouring extracts like Henna for dyeing of the hair or the skin. Excessive dermal exposure to PPD mainly leads to the N-mono- and N,N'-diacetylated products (MAPPD, DAPPD) by N-acetyltransferase 1 and 2 (NAT1 and 2) catalyzed reactions. Metabolites and PPD are mainly excreted via renal clearance. Despite a low risk of intoxication when used in due form, there are numerous cases of acute intoxication in those countries every year. At the ENT Hospital - Khartoum (Sudan) alone more than 300 cases are reported every year (~10% fatal), mostly caused by either an accidental or intended (suicidal) high systemic exposure to pure PPD. Intoxication leads to a severe clinical syndrome including laryngeal edema, rhabdomyolysis and subsequent renal failure, neurotoxicity and acute toxic hepatitis. To date, there is no defined clinical treatment or antidote available and treatment is largely supportive. Herein, we show the development of a quick on-site identification assay to facilitate differential diagnosis in the clinic and, more importantly, the implementation of an advanced analytical platform for future in-depth investigations of PPD intoxication and metabolism is described. The current work shows a sensitive (~25 µM) wet chemistry assay, a validated MALDI-MS/MS and HPLC-UV assay for the determination of PPD and its metabolites in human urine. We show the feasibility of the methods for measuring PPD over a range of 50-1000 µM. The validation criteria included linearity, lower limit of quantification (LLOQ), accuracy and precision, recovery and stability. Finally, PPD concentrations were determined in clinical urine samples of cases of acute intoxication and the applied technique was expanded to identify MAPPD and DAPPD in the identical samples.

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate--a validated assay for the clinic.

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5-1,500 ng/mL for oseltamivir and 20-1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®.