Can we accurately measure the concentration of clinically relevant vitamin D metabolites in the circulation? The problems and their consequences

Wzrost zainteresowania oznaczeniami stężenia witaminy D przyczynił się do opracowania w ostatnich latach nowych testów immunochemicznych (manualnych oraz do automatycznych analizatorów). Do laboratoriów diagnostycznych wprowadza się również techniki dotychczas niestosowane w rutynowych oznaczeniach witaminy D, takie jak HPLC (wysokosprawna chromatografia cieczowa) oraz LC-MS/MS (tandemowa spektrometria mas sprzężona z wysokosprawną chromatografią cieczową). W związku z różnorodnością testów i metod pojawia się pytanie o ich wiarygodność. W niniejszej pracy przeglądowej opisano występujące w krwiobiegu metabolity witaminy D, przedstawiamy zalety i wady stosowanych metod oznaczania ich stężenia oraz omawiamy czynniki mające wpływ na wiarygodność wyników tych oznaczeń. (Endokrynol Pol 2013; 64 (3): 238–245)Increased interest in vitamin D measurements in clinical studies has contributed to the development in recent years of several new immunochemical assays (manual and for automatic analyzers). New methods, including HPLC (high performance liquid chromatography), and LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) have also been introduced into routine diagnostic laboratories. Because of the variety of assays and methods used, the question arises which one is the most accurate for the measurement of vitamin D metabolites concentration. In this review, we summarise the advantages and disadvantages of these methods, describe the complexity of vitamin D metabolites pattern in the circulation, and discuss the problem of accurate measuring its concentration. (Endokrynol Pol 2013; 64 (3): 238–245

Czy umiemy wiarygodnie mierzyć stężenia klinicznie ważnych metabolitów witaminy D? Problemy i ich konsekwencje

Wzrost zainteresowania oznaczeniami stężenia witaminy D przyczynił się do opracowania w ostatnich latach nowych testów immunochemicznych (manualnych oraz do automatycznych analizatorów). Do laboratoriów diagnostycznych wprowadza się również techniki dotychczas niestosowane w rutynowych oznaczeniach witaminy D, takie jak HPLC (wysokosprawna chromatografia cieczowa) oraz LC-MS/MS (tandemowa spektrometria mas sprzężona z wysokosprawną chromatografią cieczową). W związku z różnorodnością testów i metod pojawia się pytanie o ich wiarygodność. W niniejszej pracy przeglądowej opisano występujące w krwiobiegu metabolity witaminy D, przedstawiono zalety i wady stosowanych metod oznaczania ich stężenia oraz omówiono czynniki mające wpływ na wiarygodność wyników tych oznaczeń

Total testosterone to dihydrotestosterone ratio assessed by LC-MS/MS predicts a worse metabolic profile not only in PCOS patients

Objectives: Total testosterone/dihydrotestosterone ratio (TT/DHT) was found to determine metabolic risk in polycystic ovary syndrome (PCOS). The aim of this study was to analyze whether (TT/DHT) may be helpful in predicting metabolic risk not only in PCOS patients but also in healthy women. Material and methods: Total testosterone (TT), dihydrotestosterone (DHT), androstendione and dehydroepiandrosterone sulphate (DHEA-S) were measured by LC-MS/MS in 36 women with PCOS and in 29 age-matched controls without clinical hyperandrogenism. In all participants, anthropometric data, lipids, adipose tissue percent (%fat), HOMA-IR were also assessed. Results: The studied groups were not different in terms of age, BMI, waist circumference, %fat and HOMA-IR. In the patients group, mean TT and androstendione levels were significantly higher as compared to controls (1.4 nmol/L vs. 1.0 nmol/L, P < 0.001) and (6.6 nmol/L vs. 4.9 nmol/L, P < 0.01), respectively. In the patients group, mean TT/DHT ratio was significantly higher compared to controls (3.6 vs. 2.7, P < 0.01) and correlated with BMI (r = 0.37, P < 0.05), waist circumference (r = 0.44, P < 0.01), %fat (r = 0.30, P < 0.05), as well as with insulin levels (r = 0.38, P < 0.05) and HOMA-IR (r = 0.44, P < 0.05). The association between TT/DHT ratio and unfavorable metabolic parameters was also seen in controls. Conclusion: Total testosterone/dihydrotestosterone ratio assessed by LC-MS/MS correlates with a worse metabolic profile not only in PCOS patients, but also in healthy women

A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium’s recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori — two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori

Ammonia Reduces Intracellular Asymmetric Dimethylarginine in Cultured Astrocytes Stimulating Its y+LAT2 Carrier-Mediated Loss

Previously we had shown that ammonia stimulates nitric oxide (NO) synthesis in astrocytes by increasing the uptake of the precursor amino acid, arginine via the heteromeric arginine/glutamine transporter y+LAT2. Ammonia also increases the concentration in the brain of the endogenous inhibitor of nitric oxide synthases (NOS), asymmetric dimethylarginine (ADMA), but distribution of ADMA surplus between the intraastrocytic and extracellular compartments of the brain has not been studied. Here we tested the hypothesis that ammonia modulates the distribution of ADMA and its analog symmetric dimethylarginine (SDMA) between the two compartments of the brain by competition with arginine for the y+LAT2 transporter. In extension of the hypothesis we analyzed the ADMA/Arg interaction in endothelial cells forming the blood-brain barrier. We measured by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) technique the concentration of arginine, ADMA and SDMA in cultured cortical astrocytes and in a rat brain endothelial cell line (RBE-4) treated with ammonia and the effect of silencing the expression of a gene coding y+LAT2. We also tested the expression of ADMA metabolism enzymes: protein arginine methyltransferase (PRMT) and dimethylarginine dimethyl aminohydrolase (DDAH) and arginine uptake to astrocytes. Treatment for 48 h with 5 mM ammonia led to an almost 50% reduction of ADMA and SDMA concentration in both cell types, and the effect in astrocytes was substantially attenuated by silencing of the Slc7a6 gene. Moreover, the y+LAT2-dependent component of ammonia-evoked arginine uptake in astrocytes was reduced in the presence of ADMA in the medium. Our results suggest that increased ADMA efflux mediated by upregulated y+LAT2 may be a mechanism by which ammonia interferes with intra-astrocytic (and possibly intra-endothelial cell) ADMA content and subsequently, NO synthesis in both cell types

Pharmacokinetic Drug Interaction Study of Sorafenib and Morphine in Rats

A combination of the tyrosine kinase inhibitor—sorafenib—and the opioid analgesic— morphine—can be found in the treatment of cancer patients. Since both are substrates of Pglycoprotein (P-gp), and sorafenib is also an inhibitor of P-gp, their co-administration may affect their pharmacokinetics, and thus the safety and efficacy of cancer therapy. Therefore, the aim of this study was to evaluate the potential pharmacokinetic drug–drug interactions between sorafenib and morphine using an animal model. The rats were divided into three groups that Received: sorafenib and morphine (ISOR+MF), sorafenib (IISOR), and morphine (IIIMF). Morphine caused a significant increase in maximum plasma concentrations (Cmax) and the area under the plasma concentration–time curves (AUC0–t, and AUC0–¥) of sorafenib by 108.3 (p = 0.003), 55.9 (p = 0.0115), and 62.7% (p = 0.0115), respectively. Also, the Cmax and AUC0–t of its active metabolite—sorafenib N-oxide—was significantly increased in the presence of morphine (p = 0.0022 and p = 0.0268, respectively). Sorafenib, in turn, caused a significant increase in the Cmax of morphine (by 0.5-fold, p = 0.0018). Moreover, in the presence of sorafenib the Cmax, AUC0–t, and AUC0–¥ of the morphine metabolite M3G increased by 112.62 (p < 0.0001), 46.82 (p = 0.0124), and 46.78% (p = 0.0121), respectively. Observed changes in sorafenib and morphine may be of clinical significance. The increased exposure to both drugs may improve the response to therapy in cancer patients, but on the other hand, increase the risk of adverse effects

In vivo assessment of potential for UGT-inhibition-based drug-drug interaction between sorafenib and tapentadol

Sorafenib (SR) is one of the most potent UGT (1A1, 1A9) inhibitors (in in vitro tests). The inhibition of UGT1A1 may cause hyperbilirubinaemia, whereas the inhibition of UGT1A9 and 1A1 may result in drug-drug interactions (DDIs). Tapentadol (TAP) is a synthetic μ-opioid agonist and is used to treat moderate to severe acute pain. Tapentadol is highly glucuronidated by the UGT1A9 and UGT2B7 isoenzymes. The aim of the study was to assess the DDI between SR and TAP. Wistar rats were divided into three groups, with eight animals in each. The rats were orally treated with SR (100 mg/kg) or TAP (4.64 mg/kg) or in combination with 100 mg/kg SOR and 4.64 TAP mg/kg. The concentrations of SR and sorafenib N-oxide, TAP and tapentadol glucuronide were respectively measured by means of high-performance liquid chromatography (HPLC) with ultraviolet detection and by means of ultra-performance liquid chromatography-tandem mass spectrometry. The co-administration of TAP with SR caused TAP maximum plasma concentration (Cmax) to increase 5.3-fold whereas its area under the plasma concentration-time curve (AUC0-∞) increased 1.5-fold. The tapentadol glucuronide Cmax increased 5.3-fold and whereas its AUC0-∞ increased 2.0-fold. The tapentadol glucuronide/TAP AUC0–∞ ratio increased 1.4-fold (p = 0.0118). TAP also increased SR Cmax 1.9-fold, whereas its AUC0-∞ increased 1.3-fold. The sorafenib N-oxide Cmax increased 1.9-fold whereas its AUC0-∞ increased 1.3-fold. The sorafenib N-oxide/SR AUC0–t ratio increased 1.4-fold (p = 0.0127). The results show that the co-administration of sorafenib and tapentadol increases the exposure to both drugs and changes their metabolism. In consequence, the pharmacological effect may be intensified, but the toxicity may increases, too.This article is published as Karbownik, Agnieszka, Miłosz Miedziaszczyk, Tomasz Grabowski, Joanna Stanisławiak-Rudowicz, Radosław Jaźwiec, Anna Wolc, Edmund Grześkowiak, and Edyta Szałek. "In vivo assessment of potential for UGT-inhibition-based drug-drug interaction between sorafenib and tapentadol." Biomedicine & Pharmacotherapy 130 (2020): 110530. doi: 10.1016/j.biopha.2020.110530.</p