A multicentric consortium study demonstrates that dimethylarginine dimethylaminohydrolase 2 is not a dimethylarginine dimethylaminohydrolase.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2's known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2.

Cerebrospinal fluid penetration of fosfomycin in patients with ventriculitis: an observational study.

BACKGROUND: For treatment of ventriculitis, vancomycin and meropenem are frequently used as empiric treatment but cerebrospinal fluid (CSF) penetration is highly variable and may result in subtherapeutic concentrations. Fosfomycin has been suggested for combination antibiotic therapy, but data are sparse, so far. Therefore, we studied CSF penetration of fosfomycin in ventriculitis. METHODS: Adult patients receiving a continuous infusion of fosfomycin (1 g/h) for the treatment of ventriculitis were included. Routine therapeutic drug monitoring (TDM) of fosfomycin in serum and CSF was performed with subsequent dose adaptions. Demographic and routine laboratory data including serum and CSF concentrations for fosfomycin were collected. Antibiotic CSF penetration ratio as well as basic pharmacokinetic parameters were investigated. RESULTS: Seventeen patients with 43 CSF/serum pairs were included. Median fosfomycin serum concentration was 200 [159-289] mg/L and the CSF concentration 99 [66-144] mg/L. Considering only the first measurements in each patient before a possible dose adaption, serum and CSF concentrations were 209 [163-438] mg/L and 104 [65-269] mg/L. Median CSF penetration was 46 [36-59]% resulting in 98% of CSF levels above the susceptibility breakpoint of 32 mg/L. CONCLUSION: Penetration of fosfomycin into the CSF is high, reliably leading to appropriate concentrations for the treatment of gram positive and negative bacteria. Moreover, continuous administration of fosfomycin appears to be a reasonable approach for antibiotic combination therapy in patients suffering from ventriculitis. Further studies are needed to evaluate the impact on outcome parameters.

Knock-out of the critical nitric oxide synthase regulator DDAH1 in mice impacts amphetamine sensitivity and dopamine metabolism.

The enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays a pivotal role in the regulation of nitric oxide levels by degrading the main endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Growing evidence highlight the potential implication of DDAH/ADMA axis in the etiopathogenesis of several neuropsychiatric and neurological disorders, yet the underlying molecular mechanisms remain elusive. In this study, we sought to investigate the role of DDAH1 in behavioral endophenotypes with neuropsychiatric relevance. To achieve this, a global DDAH1 knock-out (DDAH1-ko) mouse strain was employed. Behavioral testing and brain region-specific neurotransmitter profiling have been conducted to assess the effect of both genotype and sex. DDAH1-ko mice exhibited increased exploratory behavior toward novel objects, altered amphetamine response kinetics and decreased dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) level in the piriform cortex and striatum. Females of both genotypes showed the most robust amphetamine response. These results support the potential implication of the DDAH/ADMA pathway in central nervous system processes shaping the behavioral outcome. Yet, further experiments are required to complement the picture and define the specific brain-regions and mechanisms involved.

Simultaneous quantification of nirmatrelvir and ritonavir by LC-MS/MS in patients treated for COVID-19.

Nirmatrelvir is an antiviral agent active against SARS-CoV-2, the virus causing the pandemic disease COVID-19. It is administrated in combination with the protease inhibitor ritonavir, which acts in case of COVID-19 mainly as enzyme blocking agent preventing the premature metabolic elimination of nirmatrelvir. The combination of the two drugs in separate tablets is marketed under the brand name Paxlovid® and shows good effectivity in preventing the progression of COVID-19 to severe disease state. In this work, we described a LC-MS/MS method for the simultaneous quantification of nirmatrelvir and ritonavir in human plasma of patients treated for COVID-19 with Paxlovid®. After addition of D6-ritonavir as internal standard, plasma proteins were precipitated by the addition of methanol. The analytes were separated by gradient elution on a C18-column and were detected by tandem mass spectrometry. Calibration functions were linear in the ranges of 10 - 10000 ng/mL for nirmatrelvir and 2 - 2000 ng/mL for ritonavir. Inter-day and intra-day precision and accuracy was better than 15 % in the quality control samples and better than 20 % at the LLOQ. The method was successfully applied on samples of hospitalized patients treated for COVID-19 and proved to be capable in supporting therapeutic drug monitoring (TDM).

Overexpression of alanine-glyoxylate aminotransferase 2 protects from asymmetric dimethylarginine-induced endothelial dysfunction and aortic remodeling.

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Quantification of ceftazidime/avibactam in human plasma and dried blood spots: Implications on stability and sample transport.

Ceftazidime is an established third-generation cephalosporin antibiotic frequently administered to intensive care patients. To overcome drug resistance of pathogens, it is combined with the newly developed non-ß-lactam ß-lactamase inhibitor avibactam under the brand name Zavicefta®. To facilitate therapeutic drug monitoring (TDM), we developed a method for the simultaneous quantification of these substances by LC-MS/MS. A problem for TDM is the low stability of the analytes in plasma, requiring transport times of less than 6 h at 23 °C. Thus, we evaluated dried blood spots (DBS) as matrix for better stability. For both analytes, stable isotope labelled internal standards were applied. Plasma samples were prepared by protein precipitation, DBS by liquid extraction. The chromatographic separation took place on a polar-modified C18 column, and detection was achieved by tandem mass spectrometry with ESI ionization in positive mode for ceftazidime and negative mode for avibactam. Calibration was linear in the ranges of 5 - 100 µg/mL for ceftazidime and 1.25 - 25 µg/mL for avibactam in plasma and 2.5 - 50 µg/mL and 0.625 - 12.5 µg/mL in DBS, respectively. Precision was better than 7 % and accuracy better than 10% for plasma as well as for DBS. The stability of ceftazidime and avibactam was better in DBS than in plasma or full blood, extending maximal transport times at 23 °C from 6 h in plasma or full blood to 24 h for DBS samples. However, robust estimation of plasma concentrations from DBS measurements requires validation by future clinical studies.

Quantification of the janus kinase 1 inhibitor upadacitinib in human plasma by LC-MS/MS.

Upadacitinib is a selective janus-kinase-1 inhibitor effective in the treatment of autoimmune related diseases like rheumatoid arthritis or psoriatic arthritis. Here, we described a LC-MS/MS method for the quantification of upadacitinib in human plasma applicable for therapeutic drug monitoring. Pexidartinib was used as internal standard. Plasma samples were prepared by acidic protein precipitation and the analytes were separated on a C-18 reversed phase column. Detection took place by tandem mass spectroscopy after ionization in the positive mode and collision induced fragmentation at m/z 381 â†’ 256, 213 for upadacitinib and m/z 418 â†’ 258, 165 for pexidartinib. The calibration function was linear in the range of 0.15 - 150 ng/mL. Precisions and accuracies were better than 10% in intra- as well as inter-day evaluations. The method was applied in therapeutic drug monitoring of patients undergoing treatment for rheumatoid arthritis with the standard dose of 15 mg upadacitinib extended release formulation once daily. At steady state, we found trough levels of 4.13 (3.51 - 11.0) ng/mL, which is comparable to values found in healthy volunteers receiving the same dose (2.8 ± 1.2 ng/mL).

Fosfomycin single- and multiple-dose pharmacokinetics in patients undergoing prolonged intermittent renal replacement therapy.

BACKGROUND: Fosfomycin is used increasingly in the treatment of MDR bacteria. It is eliminated by renal excretion, but data regarding dosing recommendations for patients undergoing modern means of renal replacement therapies are scarce. OBJECTIVES: Evaluation of the pharmacokinetics (PK) of fosfomycin in patients undergoing prolonged intermittent renal replacement therapy (PIRRT) to guide dosing recommendations. METHODS: Fosfomycin was given in 11 (7 female) patients with severe infections undergoing PIRRT. Plasma levels were measured at several timepoints on the first day of fosfomycin therapy, as well as 5-6 days into therapy, before and after the dialyser, to calculate its clearance. Fosfomycin was measured in the collected spent dialysate. RESULTS: The median (IQR) plasma dialyser clearance for fosfomycin was 183.4 (156.9-214.9) mL/min, eliminating a total amount of 8834 (4556-10 440) mg of fosfomycin, i.e. 73.9% (45.3%-93.5%) of the initial dose. During PIRRT, the fosfomycin half-life was 2.5 (2.2-3.4) h. Data from multiple-dose PK showed an increase in fosfomycin Cmax from 266.8 (166.3-438.1) to 926.1 (446.8-1168.0) mg/L and AUC0-14 from 2540.5 (1815.2-3644.3) to 6714 (4060.6-10612.6) mg·h/L. Dialysis intensity during the study was 1.5 L/h. T>MIC was 100% in all patients. CONCLUSIONS: Patients undergoing PIRRT experience significant fosfomycin elimination, requiring a dose of 5 g/8 h to reach adequate plasma levels. However, drug accumulation may occur, depending on dialysis frequency and intensity.

Overexpression of dimethylarginine dimethylaminohydrolase 1 protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling.

Cardiovascular complications are the leading cause of death, and elevated levels of asymmetric dimethyarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are implicated in their pathophysiology. We investigated the role of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme hydrolyzing ADMA, in prevention of cardiovascular remodeling during hypertension. We hypothesized that the animals overexpressing DDAH1 will be protected from angiotensin II (ANG II)-induced end organ damage. Angiotensin II (ANG II) was infused in two doses: 0.75 and 1.5 mg/kg/day in DDAH1 transgenic mice (DDAH1 TG) and wild-type (WT) littermates for 2 or 4 wk. Echocardiography was performed in the first and fourth weeks of the infusion, systolic blood pressure (SBP) was measured weekly, and cardiac hypertrophy and vascular remodeling was assessed by histology. Increase in SBP after 1 wk of ANG II infusion was not different between the groups, whereas TG mice had lower SBP at later time points. TG mice were protected from cardiovascular remodeling after 2 wk of ANG II infusion in the high dose and after 4 wk in the moderate dose. TG mice had higher left ventricular lumen-to-wall ratio, lower cardiomyocyte cross-sectional area, and less interstitial fibrosis compared with WT controls. In aorta, TG mice had less adventitial fibrosis, lower medial thickness with preserved elastin content, lower counts of inflammatory cells, lower levels of active matrix metalloproteinase-2, and showed better endothelium-dependent relaxation. We demonstrated that overexpression of DDAH1 protects from ANG II-induced cardiovascular remodeling and progression of hypertension by preserving endothelial function and limiting inflammation.NEW & NOTEWORTHY We showed that overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects from angiotensin II-induced cardiovascular damage, progression of hypertension, and adverse vascular remodeling in vivo. This protective effect is associated with decreased levels of asymmetric dimethylarginine, preservation of endothelial function, inhibition of cardiovascular inflammation, and lower activity of matrix metalloproteinase-2. Our findings are highly clinically relevant, because they suggest that upregulation of DDAH1 might be a promising therapeutic approach against angiotensin II-induced end organ damage.

Homoarginine and methylarginines independently predict long-term outcome in patients presenting with suspicion of venous thromboembolism.

Endogenous arginine derivatives homoarginine, asymmetric dimethylarginine (ADMA) and symmetric dimethyarginine (SDMA) are independent mortality predictors in atherosclerotic cardiovascular disease (CVD). Our study reports the first analysis, whether homoarginine, ADMA and SDMA predict venous thromboembolism (VTE) recurrence and overall mortality in patients with suspected acute VTE. We assessed serum levels of homoarginine, ADMA and SDMA by LC-MS/MS in 865 individuals from a prospective consecutive cohort of patients with clinical suspicion of VTE. The median follow-up time for mortality was 1196 days. VTE was confirmed by imaging in 418 patients and excluded in 447 patients. Low levels of homoarginine and high levels of ADMA or SDMA independently predicted all-cause mortality after adjustment for sex, age, oral anticoagulants, body mass index, arterial hypertension, diabetes mellitus, smoking, dyslipidemia, chronic heart failure, history of stroke, creatinine and cancer both in patients with VTE and without VTE. Interestingly, none of those parameters was predictive for VTE recurrence. We provide the first report that low circulating levels of homoarginine and high circulating levels of ADMA and SDMA independently predict all-cause mortality in patients with suspected VTE. These parameters might serve as markers of "frailty" and should be considered for future risk stratification approaches in this clinical population. Taking into account that homoarginine supplementation is protective in animal models of CVD and safe in healthy human volunteers, our study provides the basis for future homoarginine supplementation studies in patients with suspected VTE to investigate possible direct protective effects of homoarginine in this population.

Clearance of chloroquine and hydroxychloroquine by the Seraph® 100 Microbinda Affinity Blood Filter -a device approved for the treatment of COVID-19 patients.

On April 17 2020, the United States Food and Drug Administration granted Coronavirus Disease 2019 (COVID-19) emergency use authorizations for the Seraph 100 Microbind Affinity Blood Filter. The medical device is aimed to treat critically ill COVID-19 patients with confirmed or imminent respiratory failure. The aim of this life size in vitro pharmacokinetic study was to investigate the in vitro adsorption of chloroquine and hydroxychloroquine from human plasma using equipment that is also used at the bedside. After start of the hemoperfusion, Pre (Cpre ) Seraph plasma levels were obtained at 5 (C5 ), 10 (C10 ), 15 (C15 ), 30 (C30 ), 60 (C60 ), and 120 (C120 ) minutes into the procedure. At two timepoints (5 and 120 minutes) post (Cpost ) Seraph plasma levels were determined that were used to calculate the plasma clearance of the Seraph. Both drugs were determined using a validated HPLC method. Median [IQR] plasma clearance of the Seraph for chloroquine/hydroxychloroquine was 1.71 [0.51-4.38] mL/min/1.79 [0.21-3.68] mL/min respectively. The lack of elimination was also confirmed by the fact that plasma levels did not change over the 120 minutes treatment. As neither chloroquine nor hydroxychloroquine were removed by the treatment with the Seraph dose adjustments in COVID-19 patients undergoing this treatment are not necessary.

The role of alanine glyoxylate transaminase-2 (agxt2) in ß-alanine and carnosine metabolism of healthy mice and humans.

PURPOSE: Chronic ß-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested ß-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to ß-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in ß-alanine homeostasis. METHODS: Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma ß-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg ß-alanine. RESULT: Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides (> 70% decrease; p < 0.05), while AGXT2 KO did not result in altered histidine-containing dipeptides levels. In both models, ß-alanine remained unaffected in the circulation and in muscle (p > 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p > 0.05). Additionally, following an acute dose of ß-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary ß-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p < 0.05). CONCLUSION: The data suggest that in vivo, ß-alanine is a substrate of AGXT2; however, its importance in the metabolism of ß-alanine and histidine-containing dipeptides seems small.

Stability of ceftolozane in human plasma and dried blood spots: Implications for transport and storage.

Ceftolozane is a newer ß-lactam antibiotic drug effective against gram-negative pathogens. Its pharmacokinetic parameters are dominated by the patients' kidney function. Consequently, in patients with impaired kidney function or those who are treated with different forms of renal replacement therapy, therapeutic drug monitoring (TDM) of ceftolozane is strongly recommended to enhance safety and efficiency of the antibiotic treatment. Various methods for the quantification of ceftolozane in plasma samples have been described utilizing HPLC-UV or LC-MS/MS. However, all these methods are impaired by the instability of ceftolozane in plasma samples. In this work, we have determined the stability of ceftolozane in human plasma at temperatures of 40 °C, 23 °C, 6 °C and - 20 °C. At 6 °C and - 20 °C, ceftolozane was stable in human plasma over the observed time range of 7 days. At 23 °C and 40 °C, plasma samples were of acceptable (i.e. less than 15% decay) stability over time ranges of 71.2 h and 5.6 h, requiring expedited sample transport to the laboratory. Dried blood spots (DBS) were reported in the literature as matrix with beneficial properties regarding stabilities of ß-lactam antibiotics. However, in this study we found that ceftolozane was of inferior stability in this matrix in comparison to plasma. Thus, DBS cannot be recommended as matrix for ceftolozane in TDM.

ADAMTS13 activity is associated with early neurological improvement in acute ischemic stroke patients treated with intravenous thrombolysis.

Although intravenous thrombolysis (IVT) with recombinant tissue-plasminogen-activator represents a highly effective treatment in acute ischemic stroke patients, not every patient benefits. We hypothesized that pretreatment levels of mediators of hemostasis (VWF and ADAMTS13) and dimethylarginines (ADMA and SDMA) are associated with early neurological improvement and outcome after IVT in ischemic stroke. Moreover we aimed to investigate the link between ADAMTS13 and markers of inflammation (CRP, IL-6, MMP-9 and MCP-1). In 43 patients with acute ischemic stroke treated with IVT blood samples for determination of the different markers were strictly taken before treatment, as well as at 24 h, 3, 7 and 90 days after symptom onset. Early neurological improvement was assessed using the shift between National Institutes of Health Stroke Scale (NIHSS) at baseline and at 24 h. Outcome at 90 days was assessed using the modified Rankin Scale. The lowest quartile of ADAMTS13 activity was independently associated with less improvement in NIHSS (baseline-24 h) (OR 1.298, p = 0.050). No independent association of ADMA or SDMA levels at baseline with outcome could be shown. Furthermore, IL-6, MCP-1 and CRP levels at 90 days significantly differed between patients with low and high ADAMTS13 activity. Thus, ADAMTS13 might indicate or even influence efficacy of IVT.

Markers of endothelial pathology to support detection of atrial fibrillation in embolic stroke of undetermined source.

A relevant part of embolic strokes of undetermined source (ESUS) is assumed to be cardiogenic. As shown previously, certain biomarkers of endothelial pathology are related to atrial fibrillation (AF). In this long-term follow-up study, we aimed to investigate whether these biomarkers are associated with subsequently diagnosed AF and with atrial cardiopathy. In 98 patients who suffered ischemic stroke of known and unknown origin L-arginine, Asymmetric (ADMA) and Symmetric Dimethylarginine (SDMA) have been measured on follow-up at least one year after index stroke. Stroke-diagnostics were available for all patients, including carotid Intima-Media-Thickness (CIMT) and comprehensive echocardiography studies. CIMT was larger in AF- compared with ESUS-patients (P < 0.001), independently from CHA2DS2VASC in the regression analysis (P = 0.004). SDMA-values were stable over time (P < 0.001; r = 0.788), whereas for ADMA moderate correlation with the initial values could be found (P = 0.007; r = 0.356). According to Kaplan-Meier-analyses, AF-detection rates were associated with CIMT (P = 0.003) and SDMA (P < 0.001). SDMA correlated with left atrial volume-index within the whole collective (P = 0.003, r = 0.322) and within the ESUS-subgroup (P = 0.003; r = 0.446). These associations were independent from CHA2DS2VASC and renal function in the regression analysis (P = 0.02 and P = 0.005, respectively). In conclusion, these results highlight SDMA and CIMT as potential markers of atrial cardiopathy and AF in ESUS-patients.

ADMA elevation does not exacerbate development of diabetic nephropathy in mice with streptozotocin-induced diabetes mellitus.

BACKGROUND AND AIMS: Cardiovascular disease is nowadays the major cause of mortality and morbidity worldwide. The risk of developing cardiovascular disease is significantly increased in patients with diabetic nephropathy. It has been suggested that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthases (NOS), may play an important role in the pathogenesis of diabetic nephropathy. ADMA is mainly metabolized by dimethylarginine dimethylaminohydrolase 1 (DDAH1). The goal of this study was to test the hypothesis that elevation of systemic ADMA levels by knocking out DDAH1 would exacerbate functional and structural glomerular abnormalities in a murine model of diabetic nephropathy. METHODS: Streptozotocin (STZ) was used to induce diabetes in adult DDAH1 knock-out and wild type mice. Healthy mice served as controls. Mice were sacrificed after 20 weeks of diabetes. Plasma ADMA levels were assessed by isotope-dilution tandem mass spectrometry and albumin by ELISA. Kidneys were used for FACS analysis and were also stained for markers of inflammation, cell proliferation, glomerular cells and cell matrix. RESULTS: STZ led to development of diabetes mellitus in all injected animals. Deficiency of DDAH1 led to a significant increase in plasma ADMA levels in healthy and diabetic mice. The diabetic state itself did not influence systemic ADMA levels. Diabetic mice of both genotypes developed albuminuria and had increased glomerulosclerosis index. There were no changes in desmin expression, glomerular cell proliferation rate, matrix expansion and expression of Mac-2 antigen in the diabetic mice of both genotypes as compared to the healthy ones. CONCLUSIONS: In summary, STZ-induced diabetes led to the development of early features of diabetic nephropathy. Deficiency of DDAH1 and subsequent increase in systemic ADMA levels did not exacerbate these changes, indicating that ADMA is not the major mediator of diabetic nephropathy in this experiment model.

The Second Life of Methylarginines as Cardiovascular Targets.

Endogenous methylarginines were proposed as cardiovascular risk factors more than two decades ago, however, so far, this knowledge has not led to the development of novel therapeutic approaches. The initial studies were primarily focused on the endogenous inhibitors of nitric oxide synthases asymmetric dimethylarginine (ADMA) and monomethylarginine (MMA) and the main enzyme regulating their clearance dimethylarginine dimethylaminohydrolase 1 (DDAH1). To date, all the screens for DDAH1 activators performed with the purified recombinant DDAH1 enzyme have not yielded any promising hits, which is probably the main reason why interest towards this research field has started to fade. The relative contribution of the second DDAH isoenzyme DDAH2 towards ADMA and MMA clearance is still a matter of controversy. ADMA, MMA and symmetric dimethylarginine (SDMA) are also metabolized by alanine: glyoxylate aminotransferase 2 (AGXT2), however, in addition to methylarginines, this enzyme also has several cardiovascular protective substrates, so the net effect of possible therapeutic targeting of AGXT2 is currently unclear. Recent studies on regulation and functions of the enzymes metabolizing methylarginines have given a second life to this research direction. Our review discusses the latest discoveries and controversies in the field and proposes novel directions for targeting methylarginines in clinical settings.