Altered Blood Molecular Markers of Cardiovascular Function in Rats after Intrauterine Hypoxia and Drug Therapy.

Many children and adults who have suffered prenatal hypoxia at an early age develop many serious diseases. This disease is an actual problem of pediatric cardiology and little studied. The aim was to analyze the cardioprotective effect of L-arginine, Thiotriazoline, Angioline, and Mildronate on the cardiovascular system of rats after prenatal hypoxia. Methods: The experiments were carried out on 50 female white rats; intraperitoneal sodium nitrite solution was administered daily to pregnant female rats after 16 days at a dose of 50 mg/kg. Control pregnant rats received saline. The offspring were divided into groups: 1-intact; 2-the control group of rat pups after PH, treated daily with physiological saline; 3-six groups of rat pups after PH, treated daily from the 1st to the 30th day after birth. Heat shock protein HSP70 was determined by enzyme immunoassay, ST2 Nitrotyrosine, and eNOS was observed by ELISA. Results: Angiolin showed a high cardioprotective effect even a month after discontinuation of the drug, and after introduction, the highest decrease in ST2 nitrotyrosine was revealed. Thiotriazoline and L-arginine have an antioxidant effect and a positive effect on eNOS expression, increasing the concentration of HSP70. Mildronate increased the expression of eNOS and the concentration of HSP70 in the blood of experimental rats after a course of administration, but did not show an antioxidant effect and did not reduce the concentration of nitrotyrosine. The results obtained indicate the cardioprotective effect of modulators of the NO system with different mechanisms of action of drugs after prenatal hypoxia.

Cardioprotective Activity of Pharmacological Agents Affecting NO Production and Bioavailability in the Early Postnatal Period after Intrauterine Hypoxia in Rats.

Intrauterine hypoxia in newborns leads to a multifaceted array of alterations that exert a detrimental impact on the cardiovascular system. The aim of this research was to assess the cardioprotective effects of modulators of the nitric oxide (NO) system, including L-arginine, Thiotriazoline, Angiolin, and Mildronate, during the early postnatal period following intrauterine hypoxia. Methods: The study involved 50 female white rats. Pregnant female rats were given a daily intraperitoneal dose of 50 mg/kg of sodium nitrite starting on the 16th day of pregnancy. A control group of pregnant rats received saline instead. The resulting offspring were divided into the following groups: Group 1-intact rats; Group 2-rat pups subjected to prenatal hypoxia (PH) and daily treated with physiological saline; and Groups 3 to 6-rat pups exposed to prenatal hypoxia and treated daily from the 1st to the 30th day after birth. Nitrotyrosine levels, eNOS, iNOS, and NO metabolites were evaluated using ELISA; to measure the expression levels of iNOS mRNA and eNOS mRNA, a PCR test was utilized. Results: Angiolin enhances the expression of eNOS mRNA and boosts eNOS activity in the myocardium of rats with ischemic conditions. Arginine and particularly Thiotriazoline exhibited a consistent impact in restoring normal parameters of the cardiac nitroxidergic system following PH. Mildronate notably raised iNOS mRNA levels and notably reduced nitrotyrosine levels, providing further support for its antioxidative characteristics.

OCTN2 enhances PGC-1α-mediated fatty acid oxidation and OXPHOS to support stemness in hepatocellular carcinoma.

BACKGROUND: The Metabolic reprogramming of tumor cells plays a vital role in the progression of hepatocellular carcinoma. Organic cation/carnitine transporter 2 (OCTN2), a sodium-ion dependent carnitine transporter and a sodium-ion independent tetraethylammonium (TEA) transporter, has been reported to contribute tumor malignancies and metabolic dysregulation in renal and esophageal carcinoma. However, the role of lipid metabolism deregulation mediated by OCTN2 in HCC cells has not been clarified. METHODS: Bioinformatics analyses and immunohistochemistry assay were employed to identify OCTN2 expression in HCC tissues. The correlation between OCTN2 expression and prognosis was elucidated through K-M survival analysis. The expression and function of OCTN2 were examined via the assays of western blotting, sphere formation, cell proliferation, migration and invasion. The mechanism of OCTN2-mediated HCC malignancies was investigated through RNA-seq and metabolomic analyses. Furthermore, xenograft tumor models based on HCC cells with different OCTN2 expression levels were conducted to analyze the tumorigenic and targetable role of OCTN2 in vivo. RESULTS: We found that gradually focused OCTN2 was significantly upregulated in HCC and tightly associated with poor prognosis. Additionally, OCTN2 upregulation promoted HCC cells proliferation and migration in vitro and augmented the growth and metastasis of HCC. Moreover, OCTN2 promoted the cancer stem-like properties of HCC by increasing fatty acid oxidation and oxidative phosphorylation. Mechanistically, PGC-1α signaling participated in the HCC cancer stem-like properties mediated by OCTN2 overexpression, which is confirmed by in vitro and in vivo analyses. Furthermore, OCTN2 upregulation may be transcriptionally activated by YY1 in HCC. Particularly, treatment with mildronate, an inhibitor of OCTN2, showed a therapeutic influence on HCC in vitro and in vivo. CONCLUSIONS: Our findings demonstrate that OCTN2 plays a critical metabolic role in HCC cancer stemness maintenance and HCC progression, providing evidence for OCTN2 as a promising target for HCC therapy.

[Clinical characteristics and metabolic therapy of fatigue in the acute and early recovery periods of ischemic stroke]. / Klinicheskie proyavleniya i metabolicheskaya terapiya astenicheskogo sindroma v ostrom i rannem vosstanovitel'nom periodakh ishemicheskogo insul'ta.

OBJECTIVE: To reveal clinical characteristics of asthenic syndrome with subsequent estimation of Meldonium therapy efficacy in patients in acute and early recovery periods of ischemic stroke. MATERIAL AND METHODS: The study included 94 patients diagnosed with ischemic stroke, mean age being 65.6±9.5 years. Psychoemotional status was assessed on the 10th day of hospitalization with the use of the Asthenic Disorders Inventory (MFI-20), Hospital Anxiety and Depression Scale (HADS), Apathy Evaluation Scale (AES). Patients with verified asthenic syndrome were added Mildronate in a daily dose of 1000 mg (500 mg 2 times a day) for one month to baseline therapy in a real outpatient setting, followed by an assessment of treatment efficacy. RESULTS: The presence of asthenic syndrome in patients in the acute period of ischemic stroke was detected on all subscales of MFI-20. A high level of general and physical asthenia was found in patients with subcortical localization stroke, with decreased motivation corresponding to lesions in the frontotemporal and decreased activity in the parieto-occipital lobes. Close correlations between the MFI-20 parameters and the level of depression, anxiety, apathy, and the degree of neurological deficit were found. The dynamic evaluation of the severity of main manifestations of psychoemotional dysfunction during treatment with Mildronate showed a decrease in the level of depression, general and mental asthenia, and decreased activity during the period of observation. CONCLUSION: The high prevalence and multifactorial character of asthenia in early ischemic stroke make it urgent to optimize the early treatment of asthenic syndrome. The results of assessment of Mildronate application in a daily dose of 1000mg for a month have shown therapy efficacy concerning affective and asthenic disorders, which allows recommending its inclusion in the complex therapy of ischemic stroke.

Mildronate Has Ameliorative Effects on the Experimental Ischemia/Reperfusion Injury Model in the Rabbit Spinal Cord.

BACKGROUND: Mildronate is a useful anti-ischemic agent and has antiinflammatory, antioxidant, and neuroprotective activities. The aim of this study is to investigate the potential neuroprotective effects of mildronate in the experimental rabbit spinal cord ischemia/reperfusion injury (SCIRI) model. METHODS: Rabbits were randomized into 5 groups of 8 animals as groups 1 (control), 2 (ischemia), 3 (vehicle), 4 (30 mg/kg methylprednisolone [MP]), and 5 (100 mg/kg mildronate). The control group underwent only laparotomy. The other groups have the spinal cord ischemia model by a 20-minute aortic occlusion just caudal to the renal artery. The malondialdehyde and catalase levels and caspase-3, myeloperoxidase, and xanthine oxidase activities were investigated. Neurologic, histopathologic, and ultrastructural evaluations were also performed. RESULTS: The serum and tissue myeloperoxidase, malondialdehyde, and caspase-3 values of the ischemia and vehicle groups were statistically significantly higher than those of the MP and mildronate groups (P < 0.001). Serum and tissue catalase values of the ischemia and vehicle groups were statistically significantly lower than those of the control, MP, and mildronate groups (P < 0.001). The histopathologic evaluation showed a statistically significantly lower score in the mildronate and MP groups than in the ischemia and vehicle groups (P < 0.001). The modified Tarlov scores of the ischemia and vehicle groups were statistically significantly lower than those of the control, MP, and mildronate groups (P < 0.001). CONCLUSIONS: This study presented the antiinflammatory, antioxidant, antiapoptotic, and neuroprotective effects of mildronate on SCIRI. Future studies will elucidate its possible use in clinical settings in SCIRI.

Neuroprotective effect of mildronate and L-carnitine on the cognitive parameters of aged mice and mice with LPS-induced inflammation.

Mildronate (MD) is a cardioprotective drug used for the treatment of cardiovascular diseases by switching metabolism from the fatty acids to glucose oxidation. This effect is achieved via inhibition of synthesis of L-carnitine (L-car), a common supplement, which is used for improving of fatty acid metabolism. Both MD and L-car have similar neuroprotective effect. Our goal was to investigate the effect of two drugs on the cognitive parameters of mice under different conditions (aging and lipopolysaccharide (LPS)-induced inflammation). We showed that L-car partly improved the memory and decreased the extent of mtDNA damage in the hippocampus of mice with the LPS-induced inflammation. L-car induced mitochondrial biogenesis and mitophagy in the Nrf2-dependent manner. Both MD and L-car upregulated expression of genes involved in the mitochondrial quality control. In 15-month-old mice, MD improved long-term and short-term memory, reduced the extent of mtDNA damage, and decreased the concentration of diene conjugates in the hippocampus in the Nrf2-independent manner. L-car as a Nrf2 activator had a better neuroprotective effect by normalizing mitochondrial quality control in the reversible cognitive impairment caused by the LPS-induced inflammation, while MD had a better neuroprotective effect in the irreversible cognitive impairment in aged mice, possibly due to a deeper restructuring of metabolism and reduction of oxidative stress.

Effect of l-carnitine and mildronate on the mitochondrial metabolism of heart and bacterial composition of the gut microbiome in ageing mice.

Ageing is the most significant risk factor for cardiovascular diseases. l-Carnitine has a potent cardioprotective effect and its synthesis decreases during ageing. At the same time, there are pharmaceuticals, such as mildronate which, on the contrary, are aimed at reducing the concentration of l-carnitine in the heart and lead to slows down the oxidation of fatty acids in mitochondria. Despite this, both l-carnitine and mildronate are positioned as cardio protectors. We showed that l-carnitine supplementation to the diet of 15-month-old mice increased expression of the PGC-1α gene, which is responsible for the regulation of fatty acid oxidation, and the Nrf2 gene, which is responsible for protecting mitochondria by regulating the expression of antioxidants and mitophagy, in the heart. Mildronate activated the expression of genes that regulate glucose metabolism. Probably, this metabolic shift may protect the mitochondria of the heart from the accumulation of acyl-carnitine, which occurs during the oxidation of fatty acids under oxygen deficiency. Both pharmaceuticals impacted the gut microbiome bacterial composition. l-Carnitine increased the level of Lachnoanaerobaculum and [Eubacterium] hallii group, mildronate increased the level of Bifidobacterium, Rikinella, Christensenellaceae. Considered, that these bacteria for protection the organism from various pathogens and chronic inflammation. Thus, we suggested that the positive effects of both drugs on the mitochondria metabolism and gut microbiome bacterial composition may contribute to the protection of the heart during ageing.

Mildronate protects heart mtDNA from oxidative stress toxicity induced by exhaustive physical exercise.

Exhaustive physical exercises are potentially dangerous for human's physical health and may lead to chronic heart disease. Therefore, individuals involved in such activity require effective and safe cardioprotectors. The goal of this research was to study Mildronate (a cardioprotective drug) effect on the level of oxidative stress markers in hearts of mice under conditions of exhausting physical exercise, such as forced swimming for 1 h per day for 7 days. Forced swimming lead to mtDNA damage accumulation, increase in diene conjugates level and loss of reduced glutathione despite an increase in antioxidant genes expression and activation of mitochondrial biogenesis. Mildronate treatment reduced oxidative stress, probably due to the inhibition of fatty acids transport to mitochondria and an increase in the intensity of glucose oxidation, which in part confirms by increase in glucose transporter expression. Thus, we can assume that Mildronate is an effective cardioprotector in exhaustive physical exercises.

Testing Meldonium: Assessing Soviet pragmatic alternatives to the randomized controlled trial.

BACKGROUND/AIMS: Current research largely tends to ignore the drug-testing model that was developed in the "Second World" as an explicit alternative to the randomized controlled trial. This system can be described as "socialist pharmapolitics," accounting for the specific features of state socialism that influenced the development and testing of experimental drugs. The clinical trials model employed in the "Second World" was heavily influenced by the Soviet Union, which was by far the most influential player in the socialist bloc during the Cold War. Based on extensive archival research, this article presents an empirical case of a late Soviet clinical trial as a pragmatic alternative to the randomized controlled trial model. It accounts for the divergences between the official model prescribed by the Soviet authorities and the messy realities of healthcare practice. It further outlines different factors that ultimately shaped how clinical trials were organized in Soviet institutions "on the ground." Accordingly, this article presents a "real-life" history of "socialist pharmapolitics" and outlines the problems that this system faced in practice. METHODS: Archival research was conducted at the Russian State Archive of Scientific and Technical Documentation in Moscow. Archival files include scientific, technical, and registration documentation such as biochemical, pharmacological, and clinical descriptions of the experimental drug Meldonium, letters between various hospitals, research institutes and the Soviet regulatory body, as well as 26 reports of completed clinical trials. Manual content analysis was used for the interpretation of results. RESULTS: This article presents an empirical case of a late Soviet clinical trial as a pragmatic alternative to the randomized controlled trial model. It demonstrates some key differences from the randomized controlled trial model. This article also highlights some of the discrepancies between the model that was officially prescribed by the Soviet authorities and the realities of experimental drug testing in the Soviet Union in the late 1980s and early 1990s. In particular, it notes some elements of randomization, double-blinding, and the use of placebo that were present in Meldonium trials despite being formally denounced by Soviet bioethics. CONCLUSION: The Soviet model for testing experimental drugs differed from the Western one substantially in a number of respects. This difference was not only proclaimed officially by the Soviet authorities, but was for the most part enforced in clinical trials in practice. At the same time, our research demonstrates that there were important differences between the official model and the clinical realities on the ground.

Neuroprotective effects of mildronate in a rat model of traumatic brain injury.

OBJECTIVE: Traumatic brain injury (TBI) is one of the most common preventable causes of mortality and morbidity. Inflammation, apoptosis, oxidative stress, and ischemia are some of the important pathophysiological mechanisms underlying neuronal loss after TBI. Mildronate is demonstrated to be beneficial in various experimental models of ischemic diseases via anti-inflammatory, antioxidant, and neuroprotective mechanisms. This study aimed to investigate possible antioxidant, anti-inflammatory, antiapoptotic, and neuroprotective effects of mildronate in a rat model of TBI. METHODS: A total of 46 male rats were divided into three groups of control, saline-treated TBI, and mildronate-treated TBI. Both TBI groups were subjected to closed-head contusive weight-drop injuries followed by treatment with saline or mildronate (100 mg/kg) administered intraperitoneally. The forebrain was removed 24 h after trauma induction, the activities of myeloperoxidase (MPO) and caspase-3, levels of superoxide dismutase (SOD), luminol- and lucigenin-enhanced chemiluminescence were measured, and histomorphological evaluation of cerebral tissues was performed. RESULTS: Increased MPO and caspase-3 activities in the vehicle-treated TBI group (p < 0.001) were suppressed in the mildronate-treated TBI group (p < 0.001). Similarly, increase in luminol and lucigenin levels (p < 0.001 and p < 0.01, respectively) in the vehicle-treated TBI group were decreased in the mildronate-treated TBI group (p < 0.001). Concomitantly, in the vehicle-treated TBI group, TBI-induced decrease in SOD activity (p < 0.01) was reversed with mildronate treatment (p < 0.05). On histopathological examination, TBI-induced damage in the cerebral cortex was lesser in the mildronate-treated TBI group than that in other groups. CONCLUSION: This study revealed for the first time that mildronate, exhibits neuroprotective effects against TBI because of its anti-inflammatory, antiapoptotic, and antioxidant activities.

Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish.

Impaired mitochondrial fatty acid ß-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid ß-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor [0.05% body weight (BW)/d] for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) ß-oxidation capability, and other molecular and biochemical assays of lipid, glucose, and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial ß-oxidation, increased the hepatic mRNA expression of genes related to FA ß-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin (mtor), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA ß-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid ß-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be used as a novel fish model for future metabolism studies.

Inhibited fatty acid ß-oxidation impairs stress resistance ability in Nile tilapia (Oreochromis niloticus).

Energy metabolism plays important roles in stress resistance and immunity in mammals, however, such functions have not been established in fish. In the present study, Nile tilapia (Oreochromis niloticus) was fed with mildronate, an inhibitor of mitochondrial fatty acid (FA) ß-oxidation, for six weeks subsequently challenged with Aeromonas hydrophila and ammonia nitrogen exposure. Mildronate treatment reduced significantly l-carnitine concentration and mitochondrial FA ß-oxidation efficiency, while it increased lipid accumulation in liver. The fish with inhibited hepatic FA catabolism had lower survival rate when exposed to Aeromonas hydrophila and ammonia nitrogen. Moreover, fish fed mildronate supplemented diet had lower immune enzymes activities and anti-inflammatory cytokine genes expressions, but had higher pro-inflammatory cytokine genes expressions. However, the oxidative stress-related biochemical indexes were not significantly affected by mildronate treatment. Taken together, inhibited mitochondrial FA ß-oxidation impaired stress resistance ability in Nile tilapia mainly through inhibiting immune functions and triggering inflammation. This is the first study showing the regulatory effects of lipid catabolism on stress resistance and immune functions in fish.

The atypical excretion profile of meldonium: Comparison of urinary detection windows after single- and multiple-dose application in healthy volunteers.

Following a one-year monitoring program providing unequivocal analytical evidence for a high prevalence in international elite sports, meldonium has been included in the World Anti-Doping Agency's (WADA) list of prohibited substances that came into effect on 1 January 2016. Despite of the polar and hydrophilic nature of the molecule, an unusual long detection window was observed in pilot elimination studies. Consequently, in the present study, urinary excretion profiles after single-dose (5 volunteers, 1×500mg) and multiple-dose oral application (5 volunteers; 2×500mg/day for 6days) were determined in order to facilitate the result management concerning meldonium findings in doping controls. Particularly the option to differentiate between recent use and tapering concentrations was studied. Urinary meldonium concentrations were determined using an analytical approach based on hydrophilic interaction liquid chromatography and high resolution tandem mass spectrometry. The study corroborates the hypothesis of a non-linear, dose-depended and biphasic excretion profile after oral application of meldonium and demonstrates that urinary detection windows are of considerable extent with up to 65 and 117days (concentrations>LOQ of 10ng/mL) following single- and multiple-dose applications, respectively.

Rapid determination of meldonium in urine samples by capillary electrophoresis with capacitively coupled contactless conductivity detection.

Capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) was employed for fast determination of meldonium (MEL) in urine samples. Background electrolyte consisting of 2M acetic acid (pH 2.3) was used for separation of MEL from cationic compounds present in urine samples and the overall analysis time was less than 4min per sample. Direct injection of urine samples was possible after 1:9 dilution with deionized water. This simple sample pretreatment was sufficient to eliminate possible matrix effects on CE performance and allowed for precise and sensitive determination of free MEL in urine. Excellent linearity (r2≥0.9998) was obtained for two concentration ranges, 0.02-4µgmL-1 and 2-200µgmL-1, by simply changing injection time from 10 to 2s without the need for additional dilution of urine samples. Limit of detection was 0.015µgmL-1 and average recoveries from urine samples spiked at 0.02-123.5µgmL-1 MEL ranged from 97.6-99.9%. Repeatability of migration times and peak areas was better than 0.35% and 4.2% for intraday and 0.95% and 4.7% for interday measurements, respectively. The above reported data proved good applicability of the CE-C4D method to determination of various MEL concentrations in urine samples and good long-term performance of the analytical system. The method might be particularly useful in analyses of large batches of samples for initial testing of MEL-positive vs. MEL-negative urine samples.

Annual banned-substance review: analytical approaches in human sports drug testing.

The aim of improving anti-doping efforts is predicated on several different pillars, including, amongst others, optimized analytical methods. These commonly result from exploiting most recent developments in analytical instrumentation as well as research data on elite athletes' physiology in general, and pharmacology, metabolism, elimination, and downstream effects of prohibited substances and methods of doping, in particular. The need for frequent and adequate adaptations of sports drug testing procedures has been incessant, largely due to the uninterrupted emergence of new chemical entities but also due to the apparent use of established or even obsolete drugs for reasons other than therapeutic means, such as assumed beneficial effects on endurance, strength, and regeneration capacities. Continuing the series of annual banned-substance reviews, literature concerning human sports drug testing published between October 2014 and September 2015 is summarized and reviewed in reference to the content of the 2015 Prohibited List as issued by the World Anti-Doping Agency (WADA), with particular emphasis on analytical approaches and their contribution to enhanced doping controls.

Establishment and application of cell models with stable expression of hOCTN1/2 / 药学学报

Human carnitine/organic cation transporter 1 and 2(hOCTN1 and hOCTN2) mediate transport of endogenous and exogenous compounds. The present study aimed to establish cell models with stable expression of hOCTN1 or hOCTN2 to study interactions with compounds and transporters. MDCK cells were transfected with pcDNA3.1(+) plasmid vector containing hOCTN1 or hOCTN2(pcDNA3.1(+)-hOCTN1/2), several stable transfected clones were obtained after G418 screening. hOCTN1 and hOCTN2 clones were screened with ergothioneine and mildronate respectively as substrates to identify the best candidates. We explored interactions of endogenous substances, alkaloids, flavonoids and ACEIs with hOCTN1/2. As a result, the cellular accumulation of ergothioneine in MDCK-hOCTN1 or mildronate in MDCK-hOCTN2 was 122 and 108 folds of the control cells, respectively. The kinetic parameters, Km and Vmax of ergothioneine, mediated by MDCK-hOCTN1, were 8.19±0.61 μmol·L-1 and 1427±49 pmol·mg-1(protein)·min-1; while Km and Vmax of mildronate by MDCKhOCTN2 were 52.3±4.3 μmol·L-1 and 2454±64 pmol·mg-1(protein)·min-1. Dopamine, glutamine, piperine, berberine, nuciferine, lisinopril and fosinopril could inhibit ergothioneine or mildronate uptake by MDCKhOCTN1/2. In conclusion, cell models with good stable hOCTN1 and hOCTN2 functions have been established successfully, which can be applied to the study of interactions between compounds and transporters of hOCTN1 and hOCTN2.

Long-chain Acylcarnitines Reduce Lung Function by Inhibiting Pulmonary Surfactant.

The role of mitochondrial energy metabolism in maintaining lung function is not understood. We previously observed reduced lung function in mice lacking the fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD). Here, we demonstrate that long-chain acylcarnitines, a class of lipids secreted by mitochondria when metabolism is inhibited, accumulate at the air-fluid interface in LCAD(-/-) lungs. Acylcarnitine accumulation is exacerbated by stress such as influenza infection or by dietary supplementation with l-carnitine. Long-chain acylcarnitines co-localize with pulmonary surfactant, a unique film of phospholipids and proteins that reduces surface tension and prevents alveolar collapse during breathing. In vitro, the long-chain species palmitoylcarnitine directly inhibits the surface adsorption of pulmonary surfactant as well as its ability to reduce surface tension. Treatment of LCAD(-/-) mice with mildronate, a drug that inhibits carnitine synthesis, eliminates acylcarnitines and improves lung function. Finally, acylcarnitines are detectable in normal human lavage fluid. Thus, long-chain acylcarnitines may represent a risk factor for lung injury in humans with dysfunctional fatty acid oxidation.

Mildronate (Meldonium) in professional sports - monitoring doping control urine samples using hydrophilic interaction liquid chromatography - high resolution/high accuracy mass spectrometry.

To date, substances such as Mildronate (Meldonium) are not on the radar of anti-doping laboratories as the compound is not explicitly classified as prohibited. However, the anti-ischemic drug Mildronate demonstrates an increase in endurance performance of athletes, improved rehabilitation after exercise, protection against stress, and enhanced activations of central nervous system (CNS) functions. In the present study, the existing evidence of Mildronate's usage in sport, which is arguably not (exclusively) based on medicinal reasons, is corroborated by unequivocal analytical data allowing the estimation of the prevalence and extent of misuse in professional sports. Such data are vital to support decision-making processes, particularly regarding the ban on drugs in sport. Due to the growing body of evidence (black market products and athlete statements) concerning its misuse in sport, adequate test methods for the reliable identification of Mildronate are required, especially since the substance has been added to the 2015 World Anti-Doping Agency (WADA) monitoring program. In the present study, two approaches were established using an in-house synthesized labelled internal standard (Mildronate-D3 ). One aimed at the implementation of the analyte into routine doping control screening methods to enable its monitoring at the lowest possible additional workload for the laboratory, and another that is appropriate for the peculiar specifics of the analyte, allowing the unequivocal confirmation of findings using hydrophilic interaction liquid chromatography-high resolution/high accuracy mass spectrometry (HILIC-HRMS). Here, according to applicable regulations in sports drug testing, a full qualitative validation was conducted. The assay demonstrated good specificity, robustness (rRT=0.3%), precision (intra-day: 7.0-8.4%; inter-day: 9.9-12.9%), excellent linearity (R>0.99) and an adequate lower limit of detection (<10 ng/mL).