In vivo profiling of phytocannabinoids in Cannabis spp. varieties via SPME-LC-MS analysis.

BACKGROUND: In vivo solid-phase microextraction (SPME) is a minimally invasive, non-exhaustive sample-preparation technique that facilitates the direct isolation of low molecular weight compounds from biological matrices in living systems. This technique is especially useful for the analysis of phytocannabinoids (PCs) in plant material, both for forensic purposes and for monitoring the PC content in growing Cannabis spp. plants. In contrast to traditional extraction techniques, in vivo SPME enables continuous tracking of the changes in the level of PCs during plant growth without the need for plant material collection. In this study, in vivo SPME utilizing biocompatible C18 probes and liquid-chromatography coupled to quadrupole time-of flight mass spectrometry (LC-Q-TOF-MS) is proposed as a novel strategy for the extraction and analysis of the acidic forms of five PCs in growing medicinal cannabis plants. RESULTS: The SPME method was optimized by testing various parameters, including the extraction phase (coating), extraction and desorption times, and the extraction temperature. The proposed method was validated with satisfactory analytical performance regarding linearity (10-3000 ng/mL), limits of quantification, and precision (relative standard deviations below 5.5 %). The proposed method was then successfully applied for the isolation of five acidic forms of PCs, which are main components of growing medicinal cannabis plants. As a proof-of-concept, SPME probes were statically inserted into the inflorescences of two varieties of Cannabis spp. plants (i.e., CBD-dominant and Δ9-THC-dominant) cultivated under controlled conditions for 30 min extraction of tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabiviarinic acid (CBVA), and tetrahydrocannabivarinic acid (THCVA). SIGNIFICANCE AND NOVELTY: The results confirmed that the developed SPME-LC-Q-TOF-MS method is a precise and efficient tool that enables direct and rapid isolation and analysis of PCs under in vivo conditions. The proposed methodology is highly appealing option for monitoring the metabolic pathways and compositions of multiple PCs in medicinal cannabis at different stages of plant growth.

Hepatocyte Nuclear Factor-1α Increases Fibrinogen Gene Expression in Liver and Plasma Fibrinogen Concentration in Rats with Experimental Chronic Renal Failure.

Chronic kidney disease (CKD) is associated with elevated plasma fibrinogen concentration. However, the underlying molecular mechanism for elevated plasma fibrinogen concentration in CKD patients has not yet been clarified. We recently found that HNF1α was significantly upregulated in the liver of chronic renal failure (CRF) rats, an experimental model of CKD in patients. Given that the promoter region of the fibrinogen gene possesses potential binding sites for HNF1α, we hypothesized that the upregulation of HNF1α can increase fibrinogen gene expression and consequently plasma fibrinogen concentration in the experimental model of CKD. Here, we found the coordinated upregulation of Aα-chain fibrinogen and Hnfα gene expression in the liver and elevated plasma fibrinogen concentrations in CRF rats, compared with pair-fed and control animals. Liver Aα-chain fibrinogen and HNF1α mRNAs levels correlated positively with (a) liver and plasma fibrinogen levels and (b) liver HNF1α protein levels. The positive correlation between (a) liver Aα-chain fibrinogen mRNA level, (b) liver Aα-chain fibrinogen level, and (c) serum markers of renal function suggest that fibrinogen gene transcription is closely related to the progression of kidney disease. Knockdown of Hnfα in the HepG2 cell line by small interfering RNA (siRNA) led to a decrease in fibrinogen mRNA levels. Clofibrate, an anti-lipidemic drug that reduces plasma fibrinogen concentration in humans, decreased both HNF1α and Aα-chain fibrinogen mRNAs levels in (a) the liver of CRF rats and (b) HepG2 cells. The obtained results suggest that (a) an elevated level of liver HNF1α can play an important role in the upregulation of fibrinogen gene expression in the liver of CRF rats, leading to an elevated concentration of plasma fibrinogen, a protein related to the risk of cardiovascular disease in CKD patients, and (b) fibrates can decrease plasma fibrinogen concentration through inhibition of HNF1α gene expression.

Hepatocyte Nuclear Factor 1α Proinflammatory Effect Linked to the Overexpression of Liver Nuclear Factor-κB in Experimental Model of Chronic Kidney Disease.

Chronic kidney disease (CKD) is associated with low-grade inflammation that activates nuclear factor-κB (NF-κB), which upregulates the expression of numerous NF-κB responsive genes, including the genes encoding IL-6, ICAM-1, VCAM-1, and MCP-1. Herein, we found the coordinated overexpression of genes encoding RelA/p65 (a subunit of NF-κB) and HNF1α in the livers of chronic renal failure (CRF) rats-an experimental model of CKD. The coordinated overexpression of RelA/p65 and HNF1α was associated with a significant increase in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. A positive correlation between liver RelA/p65 mRNA levels and a serum concentration of creatinine and BUN suggest that RelA/p65 gene transcription is tightly related to the progression of renal failure. The knockdown of HNF1α in the HepG2 cell line by siRNA led to a decrease in Rel A/p65 mRNA levels. This was associated with a decrease in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. The simultaneous repression of HNF-1α and RelA/p65 by clofibrate is tightly associated with the downregulation of IL-6, ICAM-1, VCAM-1, and MCP-1 gene expression. In conclusion, our findings suggest that NF-κB could be a downstream component of the HNF1α-initiated signaling pathway in the livers of CRF rats.

The decreased serum activity of cytosolic 5'-nucleotidase IA as a potential marker of breast cancer-associated muscle inflammation.

Cytosolic 5'-nucleotidase IA (cN-IA) plays a central role in the regulation of the purine nucleotide pool in skeletal muscle, preferentially converting adenosine monophosphate to adenosine. cN-IA can act as an autoantigen in muscle diseases, including the paraneoplastic syndrome related to breast cancer (BC). As a result of myocyte damage, released cN-IA protein may trigger the production of anti-cN-IA antibodies (anti-NT5C1A). This work aimed to develop an effective method to measure cN-IA activity in the serum and analyze it in BC patients. Our study demonstrated that serum cN-IA activity was decreased in BC patients and we assumed it is due to the presence of specific autoantibodies. We found correlations between cN-IA activity and parameters of inflammatory muscle damage. Thus, cN-IA is worth further attention to clarify its usefulness as a biomarker of BC-associated polymyositis.

An unusual nicotinamide derivative, 4-pyridone-3-carboxamide ribonucleoside (4PYR), is a novel endothelial toxin and oncometabolite.

Our recent studies identified a novel pathway of nicotinamide metabolism that involves 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) and demonstrated its endothelial cytotoxic effect. This study tested the effects of 4PYR and its metabolites in experimental models of breast cancer. Mice were divided into groups: 4T1 (injected with mammary 4T1 cancer cells), 4T1 + 4PYR (4PYR-treated 4T1 mice), and control, maintained for 2 or 21 days. Lung metastasis and endothelial function were analyzed together with blood nucleotides (including 4PYR), plasma amino acids, nicotinamide metabolites, and vascular ectoenzymes of nucleotide catabolism. 4PYR metabolism was also evaluated in cultured 4T1, MDA-MB-231, MCF-7, and T47D cells. An increase in blood 4PYR in 4T1 mice was observed at 2 days. 4PYR and its metabolites were noticed after 21 days in 4T1 only. Higher blood 4PYR was linked with more lung metastases in 4T1 + 4PYR vs. 4T1. Decreased L-arginine, higher asymmetric dimethyl-L-arginine, and higher vascular ecto-adenosine deaminase were observed in 4T1 + 4PYR vs. 4T1 and control. Vascular relaxation caused by flow-dependent endothelial activation in 4PYR-treated mice was significantly lower than in control. The permeability of 4PYR-treated endothelial cells was increased. Decreased nicotinamide but enhanced nicotinamide metabolites were noticed in 4T1 vs. control. Reduced N-methylnicotinamide and a further increase in Met2PY were observed in 4T1 + 4PYR vs. 4T1 and control. In cultured breast cancer cells, estrogen and progesterone receptor antagonists inhibited the production of 4PYR metabolites. 4PYR formation is accelerated in cancer and induces metabolic disturbances that may affect cancer progression and, especially, metastasis, probably through impaired endothelial homeostasis. 4PYR may be considered a new oncometabolite.

Comparison of different digestion methods for proteomic analysis of isolated cells and FFPE tissue samples.

Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients' treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.

APOA-1Milano muteins, orally delivered via genetically modified rice, show anti-atherogenic and anti-inflammatory properties in vitro and in Apoe-/- atherosclerotic mice.

BACKGROUND: Atherosclerosis is a slowly progressing, chronic multifactorial disease characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue that drives to the formation of asymmetric focal thickenings in the tunica intima of large and mid-sized arteries. Despite the high therapeutic potential of ApoA-1 proteins, the purification and delivery into the disordered organisms of these drugs is still limited by low efficiency in these processes. METHODS AND RESULTS: We report here a novel production and delivery system of anti-atherogenic APOA-1Milano muteins (APOA-1M) by means of genetically modified rice plants. APOA-1M, delivered as protein extracts from transgenic rice seeds, significantly reduced macrophage activation and foam cell formation in vitro in oxLDL-loaded THP-1 model. The APOA-1M delivery method and therapeutic efficacy was tested in healthy mice and in Apoe-/- mice fed with high cholesterol diet (Western Diet, WD). APOA-1M rice milk significantly reduced atherosclerotic plaque size and lipids composition in aortic sinus and aortic arch of WD-fed Apoe-/- mice as compared to wild type rice milk-treated, WD-fed Apoe-/- mice. APOA-1M rice milk also significantly reduced macrophage number in liver of WD-fed Apoe-/- mice as compared to WT rice milk treated mice. TRANSLATIONAL IMPACT: The delivery of therapeutic APOA-1M full length proteins via oral administration of rice seeds protein extracts (the 'rice milk') to the disordered organism, without any need of purification, might overcome the main APOA1-based therapies' limitations and improve the use of this molecules as therapeutic agents for cardiovascular patients.

Simultaneous overexpression of human E5NT and ENTPD1 protects porcine endothelial cells against H2O2-induced oxidative stress and cytotoxicity in vitro.

Ischemia-reperfusion injury (IRI) and oxidative stress still limit the survival of cells and organs in xenotransplantation models. Ectonucleotidases play an important role in inflammation and IRI in transplantation settings. We tested the potential protective effects derived by the co-expression of the two main vascular ectonucleotidases, ecto-5'-nucleotidase (E5NT) and ecto nucleoside triphosphate diphosphohydrolase 1 (ENTPD1), in an in vitro model of H2O2-induced oxidative stress and cytotoxicity. We produced a dicistronic plasmid (named pCX-DI-2A) for the co-expression of human E5NT and ENTPD1 by using the F2A technology. pCX-DI-2A-transfected porcine endothelial cells simultaneously overexpressed hE5NT and hENTPD1, which were correctly processed and localized on the plasma membrane. Furthermore, such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 as shown by the efficient catabolism of pro-inflammatory and pro-thrombotic extracellular adenine nucleotides along with the enhanced production of the anti-inflammatory molecule adenosine. Interestingly, we found that the hE5NT/hENTPD1 co-expression system conferred protection to cells against H2O2-induced oxidative stress and cytotoxicity. pCX-DI-2A-transfected cells showed reduced activation of caspase 3/7 and cytotoxicity than mock-, hE5NT- and hENTPD1-transfected cells. Furthermore, pCX-DI-2A-transfected cells showed decreased H2O2-induced production of ROS as compared to the other control cell lines. The cytoprotective phenotype observed in pCX-DI-2A-transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Our data add new insights to the protective effects of the combination of hE5NT and hENTPD1 against oxidative stress and constitute a proof of concept for testing this new genetic combination in pig-to-non-human primates xenotransplantation models.

Decreased serum betaine concentrations in patients after bariatric surgery.

Bariatric surgery significantly reduces the risk of cardiovascular diseases but has no effects on hyperhomocysteinemia, the risk factor for atherogenesis. We hypothesize that the decrease in serum betaine (involved in homocysteine metabolism) concentrations, after bariatric surgery, impairs conversion of homocysteine to methionine, leading to hyperhomocysteinemia. If this is true, it may be desirable to supply patients after bariatric surgery with betaine. Serum betaine and homocysteine concentrations were measured by liquid chromatography/mass spectrometry, in 16 obese patients, before and 6 months after bariatric surgery. Ten healthy individuals with normal body mass index served as controls. Serum betaine concentrations decreased to the values lower than in controls after bariatric surgery, whereas serum homocysteine concentrations remained elevated. In patients supplemented with B(12) and folate, no effect of bariatric surgery on serum concentrations of vitamins involved in homocysteine metabolism was observed. These results suggest that betaine deficit could be responsible for maintenance of hyperhomocysteinemia after bariatric surgery. We postulate that supplementation with betaine could be of therapeutic value for the treatment of hyperhomocysteinemia after bariatric surgery.

Decreased serum essential and aromatic amino acids in patients with chronic pancreatitis.

AIM: To investigate the influence of chronic pancreatitis (CP) on serum concentrations of amino acids. METHODS: Thirty-five male patients with alcoholic CP and 21 healthy male subjects were examined. Serum concentrations of amino acids were assayed by ion-pair high-performance liquid chromatography with mass detection. RESULTS: Serum glutamate concentration was increased in CP patients as compared to controls. In contrast, serum concentrations of glutamine, histidine, tyrosine, proline, tryptophan and threonine were significantly decreased in CP patients. A trend towards decreasing concentrations of serum lysine, alanine, methionine and valine as well as for total serum amino acids was observed. The sum of aromatic and the sum of essential amino acid concentrations were significantly lower in CP patients than in controls. CONCLUSION: CP leads to decreased serum concentrations of several amino acids, such as essential and aromatic serum amino acids, most likely due to decreased exocrine function.

Increased serum nitric oxide concentration after bariatric surgery--a potential mechanism for cardiovascular benefit.

BACKGROUND: It is believed that endothelial dysfunction associated with obesity contributes to reduced vascular production of nitric oxide (NO). Weight reduction after bariatric surgery is known to decrease the risk of cardiovascular disease. The purpose of this study was to determine whether bariatric surgery leads to improvement of metabolic markers of endothelial function: serum NO and its precursor (arginine) concentrations in obese patients. METHODS: Serum NO and L-arginine concentrations were measured in 25 morbidly obese patients directly before and 6 months after bariatric surgery. Moreover, selected parameters that may be involved in development of endothelial dysfunction were also studied. Control group consisted of ten healthy individuals with normal body weight. RESULTS: Six months after bariatric surgery, serum NO concentration was approximately 40% higher than before surgery. Surprisingly, serum NO concentration in nonobese controls was essentially similar to obese patients before surgery. In contrast, serum L-arginine concentration was higher in obese patients than in controls and decreased significantly after surgery. The body weight, blood pressure, triacylglycerols, LDL/HDL-cholesterol ratio, insulin, homeostasis model assessment score (HOMA-index), C-reactive protein, and white blood cell count were higher in obese patients as compared with controls and decreased significantly after surgery. CONCLUSIONS: Our results indicate that improvement of insulin resistance, lipidemia, and blood pressure as well as reduction of systemic inflammation after bariatric surgery were associated with the increase of serum NO concentration. We propose that the increase in serum NO concentration contribute to diverse beneficial effects of weight loss after bariatric surgery especially in the context of risk of atherosclerosis.

Relationship between uremic toxins and oxidative stress in patients with chronic renal failure.

OBJECTIVE: Uremic toxins play a critical role in the manifestation of the uremic syndrome. This is a consequence of retention of such substances in chronic renal failure patients and interactions between them. To date >100 uremic compounds have been discovered. The aim of this study was to elucidate potential relationships between N-methyl-2-pyridone-5-carboxamide (Me2PY) and N-methyl-4-pyridone-5-carboxamide (Me4PY), two uremic compounds, and different parameters of oxidative stress. MATERIAL AND METHODS: Forty-three non-dialyzed patients at the Nephrological Outpatients Clinic of Gdansk were enrolled and divided into two groups: (i) 20 patients with a mean estimated glomerular filtration rate (eGFR) of 22.7 ml/min/1.73 m(2); and (ii) 23 patients with a mean eGFR of 12.4 ml/min/1.73 m(2). In both groups, the plasma concentrations of uremic toxins (Me2PY, Me4PY, creatinine), malonyldialdehyde (MDA) and carbonyl groups and the erythrocyte concentration of glutathione (GSH) were analyzed. Correlations between uremic toxins and oxidative stress markers were calculated using Pearson's correlation. RESULTS: We observed significant correlations between serum creatinine and Me2PY (r=0.68; p=0.00001), eGFR and Me2PY (r=-0.55; p=0.00001), Me4PY and serum creatinine (r=0.64, p=0.00001), Me4PY and eGFR (r=-0.59; p=0.00008), MDA and Me2PY (r=0.42; p=0.006), MDA and Me4PY (r=0.38; p=0.02), GSH and Me2PY (r=-0.37; p=0.02) and GSH and Me4PY (r=-0.46; p=0.005), and in particular in patients with severe renal impairment. CONCLUSIONS: We conclude that there is a relationship between the novel uremic toxins described and oxidative stress markers. However, elucidation of the exact pathogenetic links requires further detailed studies.