Efficacy of blood plasma spectroscopy for early liver cancer diagnostics in obese patients.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) represents one of the most common cancers worldwide. A considerable proportion of HCC is caused by cirrhosis related to metabolic dysfunction-associated steatohepatitis (MASH). Due to the increasing prevalence of metabolic syndrome, it is estimated that MASH-related HCC will become the most prevalent etiology of HCC. Currently, HCC screening is based on liver ultrasonography; however, the sensitivity of ultrasonography for early HCC stages in obese patients only reaches 23 %. To date, no studied biomarker shows sufficient efficacy for screening purposes. Nevertheless, the usage of spectroscopic methods offers a new perspective, as its potential use would provide cheap, fast analysis of samples such as blood plasma. MATERIAL AND METHODS: We employed a combination of conventional and chiroptical spectroscopic methods to study differences between the blood plasma of obese cirrhotic patients with and without HCC. We included 20 subjects with HCC and 17 without evidence of liver cancer, all of them with body mass index ≥ 30. RESULTS: Sensitivities and specificities reached values as follows: 0.780 and 0.905 for infrared spectroscopy, 0.700 and 0.767 for Raman spectroscopy, 0.840 and 0.743 for electronic circular dichroism, and 0.805 and 0.923 for Raman optical activity. The final combined classification model based on all spectroscopic methods reached a sensitivity of 0.810 and a specificity of 0.857, with the highest area under the receiver operating characteristic curve among all models (0.961). CONCLUSIONS: We suggest that this approach can be used effectively as a diagnostic tool in patients who are not examinable by liver ultrasonography. CLINICAL TRIAL REGISTRATION: NCT04221347.

Untargeted metabolomics of blood plasma samples of patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths in the world. HCC is often diagnosed late because patients with early-stage cancer have no apparent symptoms. Therefore, it is desirable to find a reliable method for an early diagnosis based on the detection of metabolites - biomarkers, that can be detected in the early stages of the disease. Untargeted metabolomics is often used as a tool to find a suitable biomarker for several diseases. In this work, untargeted metabolomics was performed on blood plasma samples of HCC patients and compared with healthy individuals and patients with liver cirrhosis. A combination of liquid chromatography and high-resolution mass spectrometry was used as an analytical method. More than a thousand peaks were detected in the blood plasma samples, from which mainly amino acids, carboxylic acids, lipids, and their derivatives were evaluated as potential biomarkers. The data obtained were statistically processed using the analysis of variance, correlation analysis, and principal component analysis.

Preparations of spherical nanoparticles of chiral Cinchona alkaloid-based bridged silsesquioxanes and their use in heterogeneous catalysis of enantioselective reactions.

Two spherical nanoparticulate materials were prepared by base-catalyzed sol-gel hydrolysis/self-condensation of the bis-Cinchona alkaloid-phthalazine-based bridged bis(triethoxysilanes). For the purpose of comparing the catalytic properties, two compact materials were also prepared from the same precursors using a fluoride-catalyzed sol-gel process. All materials were characterized by SEM, TEM, solid-state 29Si NMR and 13C NMR, TGA, and FTIR. The prepared silsesquioxane-based materials were studied as potential heterogeneous catalysts for selected enantioselective reactions. The spherical material with regularly incorporated bis-quinine-phthalazine chiral units exhibited good to excellent enantioselectivities in osmium-catalyzed dihydroxylations of alkenes. Enantioselectivities observed in dihydroxylations of aromatic trans-alkenes were as excellent as those observed with the homogeneous catalyst (DHQ)2-PHAL. One compact and one nanoparticulate material was successfully recycled and reused five times without loss of enantioselectivity. Furthermore, both quinine-based and cinchonine-based materials were tested as heterogeneous organocatalysts for chlorolactonization of 4-arylpent-4-enoic acids. The materials showed only moderate enantioselectivities; however, these are the first heterogeneous catalysts for enantioselective chlorolactonization published so far.

Automated classification pipeline for real-time in vivo examination of colorectal tissue using Raman spectroscopy.

Colorectal cancer is the third most common malignancy worldwide and one of the leading causes of death in oncological patients with its diagnosis typically involving confirmation by tissue biopsy. In vivo Raman spectroscopy, an experimental diagnostic method less invasive than a biopsy, has shown great potential to discriminate between normal and cancerous tissue. However, the complex and often manual processing of Raman spectra along with the absence of a suitable instant classifier are the main obstacles to its adoption in clinical practice. This study aims to address these issues by developing a real-time automated classification pipeline coupled with a user-friendly application tailored for non-spectroscopists. First, in addition to routine colonoscopy, 377 subjects underwent in vivo acquisitions of Raman spectra of healthy tissue, adenomatous polyps, or cancerous tissue, which were conducted using a custom-made microprobe. The spectra were then loaded into the pipeline and pre-processed in several steps, including standard normal variate transformation and finite impulse response filtration. The quality of the pre-processed spectral data was checked based on their signal-to-noise ratio before the suitable spectra were decomposed and classified using a combination of principal component analysis and a support vector machine, respectively. After five-fold cross-validation, the developed classifier exhibited 100% sensitivity toward adenocarcinoma and adenomatous polyps. The overall accuracy was 96.9% and 79.2% for adenocarcinoma and adenomatous polyps respectively. In addition, an application with a graphical user interface was developed to facilitate the use of our data pipeline by medical professionals in a clinical environment. Overall, the combination of supervised and unsupervised machine learning with algorithmic pre-processing of in vivo Raman spectra appears to be a viable way of reducing the relatively large number of biopsies currently needed to definitively diagnose colorectal cancer.

Synthesis and absolute configuration of cyclic synthetic cathinones derived from α-tetralone.

The emergence of new synthetic cathinones continues to be a matter of public health concern. In fact, already known products (drugs) are being rapidly replaced by new structurally related alternatives, whereby modifications in the basic cathinone structure are used by manufacturers to circumvent the legislation. On the other hand, some derivatives of synthetic cathinones represent important pharmaceuticals with antidepressant properties. In the search for pharmaceutically relevant analogs, the main goal of the present study was to design and characterize novel cyclic α-tetralone-based derivatives of synthetic cathinones. We synthesized a series of derivatives and verified their chemical structure. Subsequently, chiral separation has been accomplished by high-performance liquid chromatography (HPLC) equipped with a circular dichroism (CD) detector, which directly provided CD spectra of the enantiomers of the analyzed substances at 252 nm. Using density functional theory calculations, we have obtained stable conformers of selected enantiomers in solution and their relative abundances, which we used to simulate their spectra. The experimental and calculated data have been used to assign the absolute configuration of six as-yet unknown synthetic cathinones.

Comprehensive spectroscopic, metabolomic, and proteomic liquid biopsy in the diagnostics of hepatocellular carcinoma.

Liquid biopsy is a very topical issue in clinical diagnostics research nowadays. In this study, we explored and compared various analytical approaches to blood plasma analysis. Finally, we proposed a comprehensive procedure, which, thanks to the utilization of multiple analytical techniques, allowed the targeting of various biomolecules in blood plasma reflecting diverse biological processes underlying disease development. The potential of such an approach, combining proteomics, metabolomics, and vibrational spectroscopy along with preceding blood plasma fractionation, was demonstrated on blood plasma samples of patients suffering from hepatocellular carcinoma in cirrhotic terrain (n = 20) and control subjects with liver cirrhosis (n = 20) as well as healthy subjects (n = 20). Most of the applied methods allowed the classification of the samples with an accuracy exceeding 80.0 % and therefore have the potential to be used as a stand-alone method in clinical diagnostics. Moreover, a final panel of 48 variables obtained by a combination of the utilized analytical methods enabled the discrimination of the hepatocellular carcinoma samples from cirrhosis with 94.3 % cross-validated accuracy. Thus, this study, although limited by the cohort size, clearly demonstrated the benefit of the multimethod approach in clinical diagnosis.

Vibrational and chiroptical analysis of blood plasma for hepatocellular carcinoma diagnostics.

More than one fifth of the world's population suffers from liver cirrhosis or other chronic liver diseases. Unfortunately, some of them will inevitably develop hepatocellular carcinoma (HCC), due to the vast majority of HCC cases arising against the background of liver cirrhosis. Despite this clearly identified high-risk group, the lack of early diagnostic options causes HCC mortality to approach its incidence. As opposed to many types of cancer, the incidence of HCC is expected to grow in the coming decades, which makes the search for an effective early diagnostic option a pressing necessity. This study presents evidence that blood plasma analysis employing a combination of chiroptical and vibrational spectroscopic methods might be the key to the improvement of the current status. One hundred samples of patients with HCC and controls with cirrhosis were classified using principal component analysis together with a random forest algorithm. Differentiation of the specific spectral patterns of the studied groups was successful in more than 80%, indicating the prospect of including spectroscopy in the screening of high-risk groups, such as patients with cirrhosis.

In vivo Raman spectroscopy in the diagnostics of colon cancer.

Early detection and accurate diagnosis of colorectal carcinoma are crucial for successful treatment, yet current methods can be invasive and even inaccurate in some cases. In this work, we present a novel approach for in vivo tissue diagnostics of colorectal carcinoma using Raman spectroscopy. This almost non-invasive technique allows for fast and accurate detection of colorectal carcinoma and its precursors, adenomatous polyps, enabling timely intervention and improved patient outcomes. Using several methods of supervised machine learning, we were able to achieve over 91% accuracy in distinguishing colorectal lesions from healthy epithelial tissue and more than 90% classification accuracy for premalignant adenomatous polyps. Moreover, our models enabled the discrimination of cancerous and precancerous lesions with a mean accuracy of almost 92%. Such results demonstrate the potential of in vivo Raman spectroscopy to become a valuable tool in the fight against colon cancer.

Prediction of Pathologic Change Development in the Pancreas Associated with Diabetes Mellitus Assessed by NMR Metabolomics.

Nuclear magnetic resonance (NMR) metabolomics was used for identification of metabolic changes in pancreatic cancer (PC) blood plasma samples when compared to healthy controls or diabetes mellitus patients. An increased number of PC samples enabled a subdivision of the group according to individual PC stages and the construction of predictive models for finer classification of at-risk individuals recruited from patients with recently diagnosed diabetes mellitus. High-performance values of orthogonal partial least squares (OPLS) discriminant analysis were found for discrimination between individual PC stages and both control groups. The discrimination between early and metastatic stages was achieved with only 71.5% accuracy. A predictive model based on discriminant analyses between individual PC stages and the diabetes mellitus group identified 12 individuals out of 59 as at-risk of development of pathological changes in the pancreas, and four of them were classified as at moderate risk.

Conformational analysis of amphetamine and methamphetamine: a comprehensive approach by vibrational and chiroptical spectroscopy.

After cannabis, the most commonly used illicit substance worldwide is amphetamine and its derivatives, such as methamphetamine, with an ever-increasing number of synthetic modifications. Thus, fast and reliable methods are needed to identify them according to their spectral patterns and structures. Here, we have investigated the use of molecular spectroscopy methods to describe the 3D structures of these substances in a solution that models the physiological environment. The substances were analyzed by Raman and infrared (IR) absorption spectroscopy and by chiroptical methods, vibrational circular dichroism (VCD) and Raman optical activity (ROA). The obtained experimental data were supported by three different computational approaches based on density functional theory (DFT) and molecular dynamics (MD). Successful interpretation relies on good agreement between experimental and predicted spectra. The determination of the conformer populations of the studied molecules was based on maximizing the similarity overlap of weighted conformer spectra by a global minimization algorithm. Very good agreement was obtained between the experimental spectra and optimized-population weighted spectra from MD, providing a detailed insight into the structure of the molecules and their interaction with the solvent. The relative population of three amphetamine and six methamphetamine conformers was determined and is consistent with a previous NMR study. However, this work shows that only a few isolated conformers are not sufficient for the successful interpretation of the spectra, but the entire conformational space needs to be sampled appropriately and explicit interaction with the solvent needs to be included.

4-Isobutylmethcathinone-A Novel Synthetic Cathinone with High In Vitro Cytotoxicity and Strong Receptor Binding Preference of Enantiomers.

New psychoactive substances and among them synthetic cathinones represent a significant threat to human health globally. However, within such a large pool of substances derived from a natural compound ((S)-cathinone), substances with important pharmaceutical uses can be identified, as already documented by bupropione. Therefore, this work aimed to find a synthetic pathway for a novel synthetic cathinone, namely 4-isobutylmethcathinone, and describe its spectroscopic properties and biological activity in vitro. Since cathinones comprise a chiral center in their structure, a method for chiral separation of the substance was elaborated using high-performance liquid chromatography on an analytical and preparative scale. Preparative enantioseparation on a polysaccharide column provided a sufficient amount of the drug for the chiroptical studies leading to the determination of the absolute configuration of enantiomers as well as for their subsequent in vitro cytotoxicity study. The cytotoxicity induced by 4-isobutylmethcathinone was determined in human cells derived from the urinary bladder (5637), neuroblastoma (SH-SY5Y), microglia (HMC-3), and hepatocellular carcinoma (Hep G2), in which the IC50 values after 72 h reached an 18-65 µM concentration. This is significantly higher cytotoxicity in comparison with other synthetic cathinones. In the receptor binding studies, a significant difference in the agonistic effect on dopamine and adrenergic receptors of individual enantiomers was observed. The lack of binding affinity towards the serotonin receptors then relates 4-isobutylmethcathinone to the family of monoamine drugs, such as 3,4-methylenedioxymathamphetamine (ecstasy, MDMA).

Comparison of genuine, generic and counterfeit Cialis tablets using vibrational spectroscopy and statistical methods.

The dubious online market in phosphodiesterase type 5 inhibitors is growing on a global scale. Counterfeit medical products can represent health issues for the user and cause medical mistrust. Within this work, genuine Cialis containing the active pharmaceutical ingredient (API) tadalafil, its generics available in the Czech Republic and the Cialis tablets from questionable online pharmacies were analysed. The methods of infra-red and Raman spectroscopy were used for the identification of the counterfeit tablets and for the verification of their API and excipients. All 9 tablets from online pharmacies were counterfeit with 2 of them even containing a different API (sildenafil, vardenafil). In addition, Raman mapping was used to determine the API and excipients' distribution and, in combination with multivariate data analysis, to separate similar tablets in clusters and to identify the outliers. Scanning electron microscopy of the samples revealed that the process of a wet granulation of micronized API was used during the formulation of the tablets. This comprehensive approach of analysis can be used for advanced exploration of the dubious samples of various medical products.

Comparison of proteomic approaches used for the detection of potential biomarkers of Alzheimer's disease in blood plasma.

At present, Alzheimer's disease is detected mainly using psychological tests, which can only confirm the disease in its more advanced phases. Therefore, bioanalytical possibilities for detecting this disease earlier are being investigated. To date, the results of analyses, which focus mainly on the study of lipids and proteins either in cerebrospinal fluid or much less often in blood plasma, do not provide satisfactory results. In addition, cerebrospinal fluid sampling is uncomfortable for the patients and involves many health risks. In this work, we deal with proteomic analysis using Matrix-Assisted Laser Desorption/Ionisation-Time of Flight and Liquid Chromatography coupled to tandem Mass Spectrometry of blood plasma with a focus on various ways of preanalytical sample treatments. This should lead to results improvement and facilitate the subsequent evaluation using principal component analysis and partial least squares discriminant analysis. The obtained results indicate the direction of further research, namely the study of interactions between proteins and lipids contained in blood plasma. These substances may be regarded as potential biomarkers allowing for the diagnosis of Alzheimer´s disease even in its early stages.

Early Detection of Pancreatic Cancer in Type 2 Diabetes Mellitus Patients Based on 1H NMR Metabolomics.

The association of pancreatic cancer with type 2 diabetes mellitus was investigated by 1H NMR metabolomic analysis of blood plasma. Concentration data of 58 metabolites enabled discrimination of pancreatic cancer (PC) patients from healthy controls (HC) and long-term type 2 diabetes mellitus (T2DM) patients. A panel of eight metabolites was proposed and successfully tested for group discrimination. Furthermore, a prediction model for the identification of at-risk individuals for future development of pancreatic cancer was built and tested on recent-onset diabetes mellitus (RODM) patients. Six of 59 RODM samples were assessed as PC with an accuracy of more than 80%. The health condition of these individuals was re-examined, and in four cases, a correlation to the prediction was found. The current health condition can be retrospectively attributed to misdiagnosed pancreatogenic diabetes or to early-stage pancreatic cancer.

Can X-Ray Powder Diffraction Be a Suitable Forensic Method for Illicit Drug Identification?

New psychoactive substances (NPSs) are associated with a significant number of intoxications. With the number of readily available forms of these drugs rising every year, there are even risks for the general public. Consequently, there is a high demand for methods sufficiently sensitive to detect NPSs in samples found at the crime scene. Infrared (IR) and Raman spectroscopies are commonly used for such detection, but they have limitations; for example, fluorescence in Raman can overlay the signal and when the sample is a mixture sometimes neither Raman nor IR is able to identify the compounds. Here, we investigate the potential of X-ray powder diffraction (XRPD) to analyse samples seized on the black market. A series of psychoactive substances (heroin, cocaine, mephedrone, ephylone, butylone, JWH-073, and naphyrone) was measured. Comparison of their diffraction patterns with those of the respective standards showed that XRPD was able to identify each of the substances. The same samples were analyzed using IR and Raman, which in both cases were not able to detect the compounds in all of the samples. These results suggest that XRPD could be a valuable addition to the range of forensic tools used to detect these compounds in illicit drug samples.

Structure of heroin in a solution revealed by chiroptical spectroscopy.

In this work, the 3-D structure of the well-known opioid drug heroin in a solution was investigated. The goal was to provide a complete and detailed description of the stable conformers with their relative abundances. This knowledge is very important from the pharmaceutical and forensic point of view as it could help significantly with deeper understanding of heroin's metabolism and the development of antagonist medicines for the case of an overdose. As heroin is a chiral compound with five stereogenic centres, the methods of chiroptical spectroscopy supplemented by density functional theory (DFT) calculations were applied to study its conformations in chloroform solution. The selected chiroptical methods, namely, electronic circular dichroism (ECD) and vibrational circular dichroism (VCD), are inherently sensitive to the 3-D structure of small- to medium-sized chiral organic molecules. A thorough conformational analysis revealed four stable conformers of heroin in chloroform solution, where the conductor-like polarizable continuum model of the solvent was used for all the calculations. The simulated ultraviolet (UV), infrared (IR), ECD, and VCD spectra were compared with the experimental ones and very good agreement was found, which enabled a detailed structure description and interpretation of the spectra. Chiroptical spectroscopy in combination with DFT calculations proved to be a very sensitive tool for the analysis of the 3-D structure of heroin in a solution in contrast with conventional spectroscopic methods. Especially, the application of VCD seems to be a promising approach for monitoring structural changes, for instance, those caused by solvents or interactions with other agents.

Plasma amyloid beta levels and platelet mitochondrial respiration in patients with Alzheimer's disease.

OBJECTIVES: Altered amyloid metabolism and mitochondrial dysfunction play key roles in the development of Alzheimer's disease (AD). We asked whether an association exists between disturbed platelet mitochondrial respiration and the plasma concentrations of Aß40 and Aß42 in patients with AD. DESIGN AND METHODS: Plasma Aß40 and Aß42 concentrations and mitochondrial respiration in intact and permeabilized platelets were measured in 50 patients with AD, 15 patients with vascular dementia and 25 control subjects. A pilot longitudinal study was performed to monitor the progression of AD in a subgroup 11 patients with AD. RESULTS: The mean Aß40, Aß42 and Aß42/Aß40 levels were not significantly altered in patients with AD compared with controls. The mitochondrial respiratory rate in intact platelets was significantly reduced in patients with AD compared to controls, particularly the basal respiratory rate, maximum respiratory capacity, and respiratory reserve; however, the flux control ratio for basal respiration was increased. A correlation between the plasma Aß42 concentration and mitochondrial respiration in both intact and permeabilized platelets differs in controls and patients with AD. CONCLUSIONS: Based on our data, (1) mitochondrial respiration in intact platelets, but not the Aß level itself, may be included in a panel of biomarkers for AD; (2) dysfunctional mitochondrial respiration in platelets is not explained by changes in plasma Aß concentrations; and (3) the association between mitochondrial respiration in platelets and plasma Aß levels differs in patients with AD and controls. The results supported the hypothesis that mitochondrial dysfunction is the primary factor contributing to the development of AD.

Blood-based molecular signature of Alzheimer's disease via spectroscopy and metabolomics.

OBJECTIVES: With over 35 million cases worldwide, Alzheimer's disease (AD) represents the main cause of dementia. The differentiation of AD from other types of dementia is challenging and its early diagnosis is complicated. The established biomarkers are not only based on the invasive collection of cerebrospinal fluid, but also lack sufficient sensitivity and specificity. Therefore, much current effort is aimed at the identification of new biomarkers of AD in peripheral blood. DESIGN AND METHODS: We focused on blood-based analyses using chiroptical spectroscopy (Raman optical activity, electronic circular dichroism) supplemented with conventional vibrational spectroscopy (infrared, Raman) and metabolomics (high-performance liquid chromatography with a high-resolution mass detection). RESULTS: This unique approach enabled us to identify the spectral pattern of AD and variations in metabolite levels. Subsequent linear discriminant analysis of the spectral data resulted in differentiation between the AD patients and control subjects. CONCLUSIONS: It may be stated that this less invasive approach has strong potential for the identification of disease-related changes within essential plasmatic biomolecules and metabolites.

Search for biomarkers of Alzheimer's disease: Recent insights, current challenges and future prospects.

Due to the trend of prolonged lifespan leading to higher incidence of age-related diseases, the demand for reliable biomarkers of dementia rises. In this review, we present novel biomarkers of high potential, especially those found in blood, urine or saliva, which could lead to a more comfortable patient experience and better time- and cost-effectivity, compared to the currently used diagnostic methods. We focus on biomarkers that might allow for the detection of Alzheimer's disease before its clinical manifestations. Such biomarkers might be helpful for better understanding the etiology of the disease and identifying its risk factors. Moreover, it could be a base for developing new treatment or at least help to prolong the presymptomatic stage in patients suffering from Alzheimer's disease. As potential candidates, we present, for instance, neurofilament light in both cerebrospinal fluid and blood plasma or amyloid ß in plasma. Above all, we provide an overview of different approaches to the diagnostics, analyzing patient's biofluids as a whole using molecular spectroscopy. Infrared and Raman spectroscopy and especially chiroptical methods provide information not only on the chemical composition, but also on molecular structure. Therefore, these techniques are promising for the diagnostics of Alzheimer's disease, as the accumulation of amyloid ß in abnormal conformation is one of the hallmarks of this disease.

Role of TiO2 Nanoparticles and UV Irradiation in the Enhancement of SERS Spectra To Improve Levamisole and Cocaine Detection on Au Substrates.

The original goal of this study was the employment of surface-enhanced Raman spectroscopy (SERS) for the analysis of real cocaine samples (containing adulterants) on composite Au-TiO2 nanomaterials to achieve low detection limits suitable for the analysis of illicit drugs and controlled substances and to exploit the photodegradation activity of TiO2 to recycle the SERS substrate for repeated analyses. The photodegradation (self-cleaning) effects of the Au-TiO2 composite nanomaterials by ultraviolet (UV) radiation are known. These effects were investigated on large-area SERS substrates immersed in the TiO2 nanoparticle aqueous suspension. The cocaine samples were measured on electrochemically gold-plated platinum targets. Surprisingly, the intensity of SERS spectra of the pure cocaine did not change after immersion in a suspension of TiO2 under UV irradiation. However, for some real cocaine samples, the overall intensity of the SERS spectra was even higher after the treatment by TiO2 and UV radiation as compared to the usual Au substrate. This unexpected signal amplification (valuable for illicit drug detection) was found to be caused mainly by the contained levamisole, which is used as a medical drug and is one of the frequent adulterants of cocaine. Both the sole effect of TiO2 on the levamisole spectrum intensity and the role of UV irradiation were inspected separately. Finally, an investigation of both the TiO2 and UV radiation treatments was performed, demonstrating (i) the necessity of both factors for selective SERS signal enhancement of the adulterant and (ii) the revision of general anticipation of the role of TiO2 in SERS systems.