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Decrease of 3-methylhistidine and increase of NG,NG-dimethylarginine in the urine of patients with muscular dystrophy
Abstract
The amounts of 3-methylhistidine, N epsilon,N epsilon-dimethyllysine, N epsilon, N epsilon, N epsilon-trimethyllysine, NG,NG-dimethylarginine, and NG,N'G-dimethylarginine were determined in the urine specimens of healthy subjects and patients of corresponding ages with Duchenne, limb-girdle, and congenital types of muscular dystrophy, and motor neuron diseases. The amount of excretion of 3-methylhistidine decreased and that of NG,NG-dimethylarginine increased significantly in Duchenne and limb-girdle types of muscular dystrophy, but not in diseases with neurogenic muscular atrophy. The decrease of 3-methylhistidine was observed consistently throughout the course of the Duchenne type of muscular dystrophy. The amounts of the other methylamino acids both in myogenic and neurogenic myopathies were not different from those in healthy subjects.
... Urine levels of ADMA (P < 0.001), DMA (P < 0.001), nitrite (P < 0.001), nitrate (P < 0.001) and hArg (P = 0.003) were significantly higher in DMD patients than in healthy controls. Regarding urinary ADMA, our observations confirm the increased urinary excretion of ADMA in DMD patients (Inoue et al. 1979). The DMA/ADMA molar ratio in urine (P = 0.002), nitrate in plasma (P < 0.001), hArg in plasma (P = 0.002) and the hArg/ADMA ratio in plasma (P < 0.001) were significantly lower in DMD patients than in healthy patients. ...
... DMD is characterized by muscular wasting and high rates of protein degradation (Inoue et al. 1979;Tran et al. 2003;Warnes et al. 1981). Protein degradation (i.e., proteolysis) is an important source of free Arg and methylarginines including ADMA (Kakimoto and Ankazawa 1970;Tran et al. 2003). ...
... However, the almost identical plasma Arg levels in DMD and healthy children argue against this possibility. Another important observation that makes proteolysis an unlikely contributor to ADMA in DMD is that the ADMA isomer symmetric dimethylarginine (SDMA, N G ,N G´dimethyl-l-arginine) was found not be elevated in urine of DMD patients (Inoue et al. 1979). As hArg is not proteinogenic, proteolysis cannot have contributed to hArg in the plasma and urine samples of our DMD patients. ...
The l-arginine/nitric oxide (L-Arg/NO) pathway regulates endothelial function and may play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). Yet, this pathway is poorly investigated in children suffering from DMD. Endothelial dysfunction can affect the perfusion of contracting muscles, thus leading to ischemia and hypoxia. In the present study, we tested the hypothesis that reduced NO production due to elevated synthesis of N
G,N
G-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of NO synthesis, is a possible pathophysiological mechanism for progressive intramuscular muscle ischemia and disturbed endothelial function in children with DMD. Given the possible antagonistic action of homoarginine (hArg) on ADMA, we also analyzed this amino acid. We investigated 55 male patients with DMD and 54 healthy male controls (HC; aged 11.9 ± 4.8 vs. 11.1 ± 4.9 years, mean ± SD). Urinary creatinine and metabolites of the L-Arg/NO pathway were measured in plasma and urine by GC–MS or GC–MS/MS. Urine levels of ADMA and its major urinary metabolite dimethylamine (DMA), nitrite and nitrate (P < 0.001 for all) and hArg (P = 0.002) were significantly higher in DMD patients compared to HC, while the urinary DMA/ADMA molar ratio was lower (P = 0.002). In plasma, nitrate (P < 0.001), hArg (P = 0.002) and the hArg/ADMA ratio (P < 0.001) were lower in DMD than in HC. In plasma, ADMA (631 ± 119 vs. 595 ± 129 nM, P = 0.149), arginine and nitrite did not differ between DMD and HC. In DMD, positive correlations between ADMA, DMA or nitrate excretion and the stage of disease (according to Vignos and Thompson) were found. In DMD patients on steroid medication, lower concentrations of ADMA in plasma, and of DMA, ADMA, nitrate and hArg in urine were observed compared to non-treated patients. The L-Arg/NO pathway is impaired in DMD patients, with the disease progression being clinically negatively correlated with the extent of impairment. One of the underlying mechanisms in DMD may involve insufficient antagonism of ADMA by hArg. Steroids, but not creatine supplementation, seems to improve the L-Arg/NO pathway in DMD.
... The levels of 3-methylhistidine urine decrease with age, whereas they tend to remain stable in healthy individuals. 108 Although metabolic biomarker candidates have been identified, it remains to not only confirm the findings and validate the most promising biomarkers, but also complement current knowledge with more targeted studies on the correlation of metabolite levels with clinical markers and disease symptoms. ...
Numerous biomarkers have been unveiled in the rapidly evolving biomarker discovery field, with an aim to improve the clinical management of disorders. In rare diseases, such as Duchenne muscular dystrophy, this endeavor has created a wealth of knowledge that, if effectively exploited, will benefit affected individuals, with respect to health care, therapy, improved quality of life and increased life expectancy. The most promising findings and molecular biomarkers are inspected in this review, with an aim to provide an overview of currently known biomarkers and the technological developments used. Biomarkers as cells, genetic variations, miRNAs, proteins, lipids and/or metabolites indicative of disease severity, progression and treatment response have the potential to improve development and approval of therapies, clinical management of DMD and patients’ life quality. We highlight the complexity of translating research results to clinical use, emphasizing the need for biomarkers, fit for purpose and describe the challenges associated with qualifying biomarkers for clinical applications.
... Abnormalities in methylarginine metabolism manifest by altered cellular, as well as tissue and urinary ADMA concentration (Tran et al. 2003). The elevated ADMA levels have been found, for example, in patients with atherosclerosis (Miyazaki et al. 1999), hypercholesterolemia , muscular dystrophy (Inoue et al. 1979) and stroke (Yoo and Lee 2001). ...
... bowel disease, 26,32 and asymmetric-dimethylarginine was elevated in urine of patients with muscular dystrophy. 33 Proliferation requires simultaneous activation of multiple pathways after oncogene activation. Amino acids, nucleic acids, polyamines, and methylation are crucial for proliferation. ...
There are no robust noninvasive methods for colorectal cancer screening and diagnosis. Metabolomic and gene expression analyses of urine and tissue samples from mice and humans were used to identify markers of colorectal carcinogenesis.
Mass spectrometry-based metabolomic analyses of urine and tissues from wild-type C57BL/6J and Apc(Min/+) mice, as well as from mice with azoxymethane-induced tumors, was employed in tandem with gene expression analysis. Metabolomics profiles were also determined on colon tumor and adjacent non-tumor tissues from 39 patients. The effects of β-catenin activity on metabolic profiles were assessed in mice with colon-specific disruption of Apc.
Thirteen markers were found in urine associated with development of colorectal tumors in Apc(Min/+) mice. Metabolites related to polyamine metabolism, nucleic acid metabolism, and methylation, identified tumor-bearing mice with 100% accuracy, and also accurately identified mice with polyps. Changes in gene expression in tumor samples from mice reflected the observed changes in metabolic products detected in urine; similar changes were observed in mice with azoxymethane-induced tumors and mice with colon-specific activation of β-catenin. The metabolic alterations indicated by markers in urine therefore appear to occur during early stages of tumorigenesis, when cancer cells are proliferating. In tissues from patients, tumors had stage-dependent increases in 12 metabolites associated with the same metabolic pathways identified in mice (including amino acid metabolism and polyamine metabolism). Ten metabolites that were increased in tumor tissues, compared with non-tumor tissues (proline, threonine, glutamic acid, arginine, N1-acetylspermidine, xanthine, uracil, betaine, symmetric dimethylarginine, and asymmetric-dimethylarginine), were also increased in urine from tumor-bearing mice.
Gene expression and metabolomic profiles of urine and tissue samples from mice with colorectal tumors and of colorectal tumor samples from patients revealed metabolites associated with specific metabolic changes that are indicative of early-stage tumor development. These urine and tissue markers might be used in early detection of colorectal cancer.
... Muscular dystrophy is a disease characterized by muscular wasting and rapid degradation of all classes of muscle protein. In patients with this disease, increased urinary ADMA levels have been reported with no change in SDMA levels compared with healthy controls [14]. Increased protein degradation has also been reported in cardiac ischemia [15] and diabetes [16]. ...
In this study, we examined changes in asymmetric dimethylarginine (ADMA), dimethylarginine dimethylaminohydrolase (DDAH), nitric oxide synthesis (NOS), and the arginine methylation of organ proteins during the development of diabetes in mice.
Db/db mice developed significant obesity and fasting hyperglycemia during diabetogenesis. During diabetogenesis, the expression of ADMA and nNOS was increased, while that of DDAH1 and protein-arginine methyltransferase 1 (PRMT1) was decreased. Additionally, arginine methylation in the liver and adipose tissue was altered during diabetogenesis.
Changes were evident at 75, 60, and 52 kDa in liver tissue and at 38 and 25 kDa in adipose tissue. Collectively, DDAH and ADMA are closely associated with the development of obesity and diabetes, and the arginine methylation levels of certain proteins were changed during diabetes development.
Protein arginine methylation plays a role in the pathogenesis of diabetes.
... Another possible explanation for the predictive power of SDMA in terms of mortality is the fact that SDMA is increased in states of high protein turnover (41,42 ). Protein catabolism as part of the metabolic syndrome is associated with increased mortality, and this could help to explain the predictive power of SDMA. ...
Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, has been linked to cardiovascular risk. The clinical role of its structural isomer symmetrical dimethylarginine (SDMA) remains largely unclear.
We measured SDMA and ADMA in 3229 patients undergoing coronary angiography at baseline (1997-2000) and recorded total and cardiovascular mortality during a median follow-up time of 7.7 years. We investigated associations of SDMA with cardiovascular risk factors and mortality and compared its role as a cardiovascular risk factor with ADMA, which predicted mortality in previous analyses of our study.
In linear regression analyses including common cardiovascular risk factors as covariates, SDMA and ADMA were significantly associated with cystatin C, N-terminal pro-B-type natriuretic peptide, New York Heart Association classification, and homocysteine. The regression coefficients were higher for SDMA than for ADMA. In Cox proportional-hazards models adjusted for cardiovascular risk factors, the hazard ratios (HRs) (with 95% CI) in the second, third, and fourth SDMA quartile compared to the lowest quartile were 0.77 (0.60-0.99), 0.99 (0.78-1.25), and 1.51 (1.20-1.91) for total mortality and 0.92 (0.68-1.25), 0.93 (0.68-1.26), and 1.54 (1.14-2.01) for cardiovascular mortality. The same calculations for ADMA quartiles revealed HRs of 1.05 (0.83-1.32), 1.19 (0.95-1.50), and 1.61 (1.30-1.99) for total mortality and HR of 1.00 (0.74-1.34), 1.26 (0.95-1.68), and 1.54 (1.18-2.02) for cardiovascular mortality.
Serum concentrations of SDMA are independently associated with increased cardiovascular and all-cause mortality in patients undergoing coronary angiography. The pattern of risk linked to SDMA is different from that linked to ADMA, suggesting different pathophysiological roles of these 2 methylarginine metabolites.
... Abnormalities in methylarginine metabolism manifest by altered cellular, as well as tissue and urinary ADMA concentration (Tran et al. 2003). The elevated ADMA levels have been found, for example, in patients with atherosclerosis (Miyazaki et al. 1999), hypercholesterolemia , muscular dystrophy (Inoue et al. 1979) and stroke (Yoo and Lee 2001). ...
Protein arginine methylation represents a posttranslational modification undertaken by protein arginine methyltransferases (PRMT) that results in production of protein-incorporated omega-NG-monomethylarginine (L-NMMA), asymmetric omega-NG, NG-dimethylarginine (ADMA), or omega-NG, N G-dimethylarginine (SDMA). Free cellular L-NMMA, ADMA and SDMA can be generated via the proteolytic cleavage of intracellular proteins, thereby also affecting methylarginine content in the plasma. Free methylarginines can be cleared from the body by renal excretion. L-NMMA and ADMA, but not SDMA, can be degraded via enzymes called NG, NGdimmethylarginine dimethylaminohydrolase (DDAH).
ADMA is an endogenous inhibitor of nitric oxide synthases (NOS) and a marker of endothelial dysfunction. Increased plasma ADMA levels have been reported in patients with cardiovascular disorders including pulmonary arterial hypertension (PAH), a fatal disease characterized by elevated blood pressure in the pulmonary circulation, due to increased resistance of pulmonary arterioles. The major pathophysiologic hallmark of PAH is pulmonary arterial smooth muscle cell (PASMC) hypertrophy and proliferation, leading to the occlusion of pulmonary arterioles. The interplay between methylarginine synthesis and degradation in vivo, as well as specific alterations to intrapulmonary ADMA levels or distorted generation of ADMA in PAH, however, remains to be elucidated.
In the current study, we hypothesized that methylarginine production and degradation is tissue-specific and that the lung has a significant impact on serum/plasma ADMA levels, possibly leading to endothelial dysfunction observed in PAH. To this end, we sought to address the following specific aims: 1) to develop a novel, HPLC-based method to assess protein-incorporated and free cellular methylarginine content in biological samples, 2) to analyze the tissue-specific methylarginine metabolism in normal subjects, and 3) to analyze the methylarginine content in the lungs of patients with PAH compared with healthy donors.
First, to analyze tissue-specific methylarginine metabolism in the normal physiological state, we performed high performance liquid chromatography (HPLC)-driven assessment of protein-incorporated and free cellular methylarginine levels, together with Western blot analyses of PRMT and DDAH expression, in organs of the cardiovascular system. Our results revealed that pulmonary expression of type I PRMT was correlated with enhanced protein arginine methylation in the lung. Moreover, our studies also revealed that the kidney and the liver provide complementary routes for clearance and metabolic conversion of circulating ADMA.
To address the impact of intrapulmonary ADMA metabolism in pathogenic conditions, we next analyzed lung homogenates of PAH patients. HPLC analysis revealed significantly lower levels of protein-incorporated ADMA in the lungs of PAH patients (n=12), compared with controls (n=10, transplant donors). Western Blot analyses confirmed a significantly decreased content of asymmetrically dimethylated proteins in PAH lungs. The expression of PRMT, in particular PRMT1, was decreased in PAH. Immunohistochemical staining of IPAH and control lungs localized PRMT1 to pulmonary arterial vascular smooth muscle cells (PASMC). Moreover, PRMT1 knockdown in primary PASMC by siRNA technology significantly increased PASMC proliferation.
Our results demonstrate that, in the normal physiological state, methylarginine metabolism by the pulmonary system significantly contributes to circulating methylarginine levels. In pathogenic conditions, protein-incorporated ADMA concentrations do not reflect free cellular levels of ADMA in the lung. This may be explained by the alterations of DDAH activity in the lung, which, consequently, regulate ADMA content in the serum of IPAH patients. In addition, our studies demonstrated a novel regulatory role of PRMT1 in progression of PAH, by the alteration of PASMC proliferation, a major characteristic of PAH. This led to conclusions that protein arginine methylation plays a pivotal role in the pathogenesis of PAH. Posttranslationale Protein-Arginin Methylierung erfolgt durch eine Gruppe spezifischer Protein-Arginin Methyltransferasen (PRMTs), die neben der Bildung von asymmetrischem Dimethylarginin (ADMA) auch für die Synthese von Monomethylarginin (L-NMMA) und symmetrischem Dimethylarginin (SDMA) verantwortlich sind. Die Freisetzung von Methylarginine in das Blutplasma erfolgt nach heutigem Wissensstand über die Proteolyse zellulärer, methylierter Proteine. Alle Methylargininformen werden über renale Exkretion aus dem Körper eliminiert. Neuere Studien heben die Metabolisierung von ADMA und L-NMMA durch das Enzym Dimethylarginin-Dimethylaminohydrolase (DDAH) als Hauptabbauweg hervor.
ADMA ist ein endogener Inhibitor der NO-Synthase und ein Marker für endotheliale Fehlfunktion. Eine erhöhte ADMA Konzentration im Blut wird bei verschiedenen kardiovaskulären Erkrankungen, so auch bei pulmonal-arterieller Hypertonie (PAH), für einen Mangel an biologisch verfügbarem NO verantwortlich gemacht. Die pulmonal-arterielle Hypertonie ist durch eine pathologische Hypertrophie und Proliferation pulmonalarterieller glatter Muskelzellen (PASMC) gekennzeichnet, die eine Okklusion pulmonaler Arteriolen zur Folge hat. Ob ein Zusammenhang zwischen Arginin- und Dimethylargininstoffwechsel und den bei PAH zu beobachtenden Symptomen vorliegt, wurde bislang nicht untersucht.
Deshalb sollte in der vorliegenden Studie geprüft werden, ob der Methylarginin-metabolismus der Lunge signifikant zur ADMA Konzentration im Blut beiträgt und somit an der Ausbildung endothelialer Fehlfunktionen beteiligt sein könnte. Im Konkreten sollten hierfür folgende Vorhaben realisiert werden: (1) Entwicklung einer auf Hochdruckflüssigkeitschromatographie-basierenden Methode zur Quantifizierung von protein-inkorporiertem und freiem Methylarginin in biologischen Proben, (2) Analyse des gewebespezifischen Methylargininmetabolismus und (3) Bestimmung des pulmonalen Methylarginingehaltes von PAH Patienten und gesunden Organspendern.
Zur Beschreibung des Methylargininmetabolismus unter normalen physiologischen Bedingungen wurden protein-inkorporiertes und freies Methylarginin in Organen des kardiovaskulären Systems bestimmt. Zudem wurde vergleichend Proteinexpression und Aktivität der PRMTs und DDAHs ermittelt. Unsere Untersuchungen ergaben eine klare Korrelation zwischen pulmonaler Typ I PRMT Proteinexpression und erhöhter Protein-Arginin Methylierung. Zudem konnte gezeigt werden, dass Niere und Leber komplementär an der Eliminierung und Metabolisierung von ADMA und L-NMMA beteiligt sind.
Zur Beurteilung der Frage, ob bei PAH ein geänderter Dimethylargininstoffwechsel zu beobachten ist, wurde Lungenhomogenat mittels HPLC untersucht. Die Analyse bei PAH Patienten (n=12) und gesunden Organspendern (n=10) ergab eine signifikante Abnahme an protein-inkorporiertem ADMA bei PAH Patienten. Zudem konnte über Western-Blot Analyse ein reduzierter Gehalt an asymmetrisch dimethylierten Proteinen nachgewiesen werden. Bei PAH Patienten zeigte sich auch eine signifikant reduzierte Expression jener Protein-Arginin-Methyltransferasen, insbesondere PRMT 1, die für eine asymmetrische Dimethylierung von Zielproteinen verantwortlich sind. Immunohistochemische Untersuchungen führten zu dem Ergebnis, dass PRMT 1 überwiegend in PASMCs lokalisiert ist. Zudem resultierte die Reduktion der PRMT 1 Expression mittels siRNA Technologie in einer Zunahme der PASMC Proliferation.
Aus den vorliegenden Ergebnissen lässt sich somit schlussfolgern, dass der pulmonale Dimethylargininmetabolismus maßgeblich zum Plasma ADMA-Spiegel beiträgt. Bei PAH Patienten konnte keine Korrelation zwischen protein-inkorporiertem ADMA und freiem Methylarginin nachgewiesen werden. Dieses Ergebnis deutet auf eine Änderung der pulmonalen DDAH Aktivität und Plasma ADMA-Werte bei PAH Patienten hin. Des Weiteren konnte eindeutig demonstriert werden, dass PRMT 1 an der Regulation der PASMC Proliferation beteiligt ist. Zusammenfassend lässt sich somit feststellen, dass Protein-Arginin Methylierung an der Entwicklung und am Fortschreiten von PAH beteiligt sein könnte.
... Here, we extend these findings to human populations with and without pathophysiologic levels in plasma as an initial step to characterize DMA stores in circulation. In support of these findings, other groups have reported a correlation between elevated plasma ADMA levels in conditions of high protein turnover such as in nephrotic syn- drome [38], metabolic syndrome [18] and muscular dys- trophy [39]. In our study, the ESRD population, known to have the highest levels of plasma ADMA, demonstrated the capacity to accumulate ADMA in greater amounts and at faster rates than similar subjects without renal impairment (P = 0.03), suggesting a source for an ongoing ADMA burden . ...
... The enzyme necessary for formation of ADMA exists in the central nervous system [209][210][211]. This suggests that central neural mechanisms may be involved in systemic responses to NOS inhibitors in diseases such as renal failure [50], muscle dystrophy [212,213], hypercholesterolaemia [57] and pregnancy with eclampsia [214], premature infants [215], where raised plasma or urine ADMA levels have been demonstrated. It is also possible that NO modulates peripheral sympathetic neurotransmission [216] and NO directly released from nerves alters vascular tone [217]. ...
Vascular endothelium releases nitric oxide (NO), an important vasodilator that is continuously synthesised by the constitutive enzyme, endothelial nitric oxide synthase (NOS). This maintains a constant vasodilator tone which is diminished in adult hypertension, due to reduced endothelium-dependent vascular relaxation, which is NO dependent. In childhood, however, hypertension is often secondary, and normalisation of blood pressure by removal of cause (e.g. renal artery stenosis, catecholamine-producing tumour) suggests reversibility of endothelial dysfunction, if it is present. Raised plasma levels of endogenous inhibitors have been found, especially in children with secondary hypertension due to renal parenchymal and renovascular disease, and may contribute to hypertension by more than just inhibition of vascular NO release; e.g. by reduction of glomerular filtration rate and promotion of salt and water retention. These inhibitors also modulate renin release, which may be of relevance in cardiovascular physiology, and may also interfere with the anti-platelet properties of NO, increasing the likelihood of vascular thrombotic events. NO inhibitors also promote endothelial activation, with increased expression of adhesion molecules that may form seedlings of atherosclerosis. In chronic renal impairment, accumulation of NO inhibitors may contribute to hypertension. Efficient long-session dialysis helps better interdialysis control of blood pressure in these subjects, independent of salt and water removal, suggesting that removal of such vasoactive agents may be important for efficient blood pressure control. There are a few studies assessing NO generation in hypertensive children via plasma nitrite and nitrate, the NO end products, which suggest normal or increased production as opposed to a reduction, perhaps as a compensatory phenomenon. In the treatment of hypertension, nitroprusside and nitrates exert their actions via NO donation. Excessive production of NO (usually via inducible NOS) or excessive administration (nitrovasodilators) can be cytotoxic and may cause hypotension and shock, as in severe sepsis. NOS inhibitors and NO therefore appear to play a crucial role in aetiology, complications and therapy of childhood hypertension.
... We were unable to find published data that assessed MA and protein kinetics in the same subjects. Elevated plasma levels of MA have been predicted to be associated with states of increased protein breakdown due to a variety of 'stress' states [9], but without supportive human data other than increased renal ADMA excretion in muscular dystrophy [36]. The rates of release of NMMA, ADMA and SDMA from methylated proteins depend on the rates of protein catabolism. ...
Increased circulating methylarginines (MA) have been linked to the metabolic syndrome to explain endothelial dysfunction and cardiovascular disease risk. Proteins that contain MA are regulatory and release them during catabolism. We hypothesised that increased protein turnover in insulin-resistant states contributes to an increase in circulating MA. MATWERIALS AND METHODS: We performed hyperinsulinaemic, euglycaemic, and isoaminoacidaemic experiments on 49 lean, obese and elderly subjects, with measurements of the kinetics of glucose and protein metabolism. Plasma MA, i.e. asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), and N -monomethyl-L-arginine (NMMA), lipids and body composition were measured.
Insulin resistance of glucose and protein metabolism occurred in obese and elderly subjects. ADMA concentrations were 29 to 120% higher in obese and 34% higher in elderly than in lean subjects. SDMA were 34 and 20% higher in obese than in lean and than in elderly subjects, respectively. NMMA were 32% higher in obese than in lean subjects. ADMA differed by sex, being higher in men, namely by 1.75x in obese men and by 1.27x in elderly men. Postabsorptive ADMA (r=0.71), SDMA (r=0.46), and NMMA (r=0.31) correlated (all p<0.05) with rates of protein flux. All three MA correlated negatively with clamp glucose infusion rates and uptake (p<0.001). ADMA and SDMA correlated negatively with net protein synthesis and clamp amino acid infusion rates (p<0.05). All MA also correlated with adiposity indices and fasting insulin and triglycerides (p<0.05).
Obesity, sex and ageing affect MA. Elevations of the three MA in obese, and of ADMA in elderly men, are related to increased protein turnover and to lesser insulin sensitivity of protein metabolism. These interrelationships might amplify insulin resistance and endothelial dysfunction.
... If hemolysis and subsequent protein turnover do contribute to ADMA accumulation in WB, it is likely that pathological conditions characterized by RBC fragility or enhanced expression of proteolytic enzymes may facilitate increases in free plasma ADMA concentrations. The hypothesis that RBC rheology and altered protein turnover may have roles in ADMA release is supported by recent publications showing increased plasma ADMA in patients with sickle cell anemia (47), a correlation between ADMA and RBC fragility in hypertensive subjects (57), and elevated ADMA levels in the urine of patients afflicted with muscular dystrophy (21). As a further contributor to endothelial dysfunction, it is notable that RBCs contain significant quantities of arginase, an enzyme that hydrolyzes L-arginine to urea and ornithine. ...
The endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is elevated in many patients and may contribute to the initiation and progression of their disease. While some mechanistic pathways have been identified, tissue-specific contributions to ADMA control remain unclear. We sought to determine if whole blood (WB) could participate in ADMA control ex vivo. Anesthetized male Sprague-Dawley rats underwent exsanguinations, and WB preparations were incubated at 37 degrees C for 5 h. ADMA and symmetrical dimethylarginine were analyzed by high-pressure liquid chromatography. Incubation of lysed red blood cell (RBC) supernatant yielded a significant decrease in ADMA that was blocked by 4124W, a synthetic inhibitor of dimethylarginine dimethylaminohydrolase, the only reported enzyme to hydrolyze ADMA. Hydrolysis of ADMA was diminished by addition of physiologically relevant concentrations of zinc (i.e., 20 microM). Conversely, when rat WB or WB supernatant was incubated at 37 degrees C, it liberated quantities of free ADMA (1-2 microM) that in vivo would likely have pathological consequences. Addition of arginine methyltransferase inhibitors to these incubations did not reduce ADMA release, indicating no dominant role for active protein methylation during these incubations. This ADMA liberation was significantly reduced by addition of protease inhibitors, indicating a dependence on peptide bond hydrolysis. Total ADMA (protein incorporated plus free) was determined by acid hydrolysis and found to be 43.18 +/- 4.79 microM in WB with approximately 95% of this in RBCs. These ex vivo data demonstrate the potential of blood to control the NO-NOS system by modulating free ADMA.
... Protein methylation is augmented in proliferating cells and, indeed, ADMA concentration is higher in regenerating than in quiescent endothelial cells [7]. Finally, anti-DNA antibodies stimulate methylation of ribonucleoproteins and may underlie increased synthesis of methylarginines in patients with systemic lupus [26,149] The increased proteolysis may contribute to elevation of ADMA in hypercatabolic states such as endotoxemia [109], hyperthyroidism [58] and muscular dystrophy [62,97]. Impaired urinary excretion leads to overaccumulation of methylarginines in uremic patients [50], but may also contribute to elevation of ADMA in hemorrhagic [5] or septic shock [114]. ...
Asymmetric dimethylarginine (ADMA) is synthesized during the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and is released during proteolysis. ADMA is a competitive inhibitor of nitric oxide synthase and may decrease NO availability. ADMA is eliminated by renal excretion or is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) to citruline and dimethylamine. Two other endogenous methylarginines are also synthesized by PRMT: N-monomethyl-L-arginine (L-NMMA) and symmetric dimethylarginine (SDMA). L-NMMA inhibits NO synthase but its concentrations in circulation are much lower than ADMA whereas SDMA is inactive. Plasma concentration of ADMA is markedly increased in patients with chronic renal failure and moderately increased in patients with many other diseases including hyperlipidemia, diabetes mellitus, arterial hypertension, hyperhomocysteinemia and heart failure. The increased concentration of ADMA is positively correlated with markers of atherosclerosis, such as carotid artery intima-media thickness and has a predictive value for acute cardiovascular events in prospective studies. Angiotensin-converting enzyme inhibitors, angiotensin AT1 receptor antagonists, vitamin E and, according to some studies, estrogens used in hormonal replacement therapy reduce plasma ADMA concentration, which may contribute to their beneficial effect on NO synthesis and endothelial function. However, in some states associated with excess of NO, such as septic shock or excitotoxic neuronal injury ADMA may be protective by limiting toxic effect of high concentrations of NO. This article reviews the effect of pharmacotherapy on ADMA metabolism and its possible clinical implications.
... Upon proteolysis, which is increased in hyper metabolic states such as cirrhosis and inflammation, 31 significant amounts of ADMA are generated. 32 The rate of ADMA and SDMA generation is thus likely to be dependent on the presence and activity of PRMTs and the rate of protein breakdown. In keeping with this, we observed an increase in expression of PRMT-1 in AHϩCϩ patients in whom there is increased inflammation, compared to AH-Cϩ patients. ...
Unlabelled:
Previous studies suggest reduced hepatic endothelial nitric oxide synthase activity contributes to increased intrahepatic resistance. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, undergoes hepatic metabolism via dimethylarginine-dimethylamino-hydrolase, and is derived by the action of protein-arginine-methyltransferases. Our study assessed whether ADMA, and its stereo-isomer symmetric dimethylarginine (SDMA), are increased in alcoholic hepatitis patients, and determined any relationship with severity of portal hypertension (hepatic venous pressure gradient measurement) and outcome. Fifty-two patients with decompensated alcoholic cirrhosis were studied, 27 with acute alcoholic hepatitis and cirrhosis, in whom hepatic venous pressure gradient was higher (P = 0.001) than cirrhosis alone, and correlated with ADMA measurement. Plasma ADMA and SDMA were significantly higher in alcoholic hepatitis patients and in nonsurvivors. Dimethylarginine-dimethylamino-hydrolase protein expression was reduced and protein-arginine-methyltransferase-1 increased in alcoholic hepatitis livers. ADMA, SDMA and their combined sum, which we termed a dimethylarginine score, were better predictors of outcome compared with Pugh score, MELD and Maddrey's discriminant-function.
Conclusion:
Alcoholic hepatitis patients have higher portal pressures associated with increased ADMA, which may result from both decreased breakdown (decreased hepatic dimethylarginine-dimethylamino-hydrolase) and/or increased production. Elevated dimethylarginines may serve as important biological markers of deleterious outcome in alcoholic hepatitis.
Duchenne muscular dystrophy is a severe, X-linked disease characterized by decreased muscle mass and function in children. Genetic and biochemical research over the years has led to the characterization of the cause and the pathophysiology of the disease. Moreover, the elucidation of genetic mechanisms underlining Duchenne muscular dystrophy has allowed for the design of innovative personalized therapies.
The identification of specific, accurate, and sensitive biomarkers is becoming crucial for evaluating muscle disease progression and response to therapies, disease monitoring, and the acceleration of drug development and related regulatory processes.
This review illustrated the up-to-date progress in the development of candidate biomarkers in DMD at the level of proteins, metabolites, micro-RNAs (miRNAs) and genetic modifiers also highlighting the complexity of translating research results to clinical practice.
We highlighted the challenges encountered in translating biomarkers into the clinical context and the existing bottlenecks hampering the adoption of biomarkers as surrogate endpoints. These challenges could be overcome by national and international collaborative efforts, multicenter data sharing, definition of public biobanks and patients’ registries, and creation of large cohorts of patients. Novel statistical tools/ models suitable to analyze small patient numbers are also required.
Finally, collaborations with pharmaceutical companies would greatly benefit biomarker discovery and their translation in clinical trials.
The dystrophin-glycoprotein complex (DGC) is a transmembrane structure that links the cytoskeleton of muscle cells to the extracellular matrix. Genetic disruption of this complex in muscular dystrophies causes sarcolemmal instability that results in injury and death of the muscle cells and causes altered activation of mechanosensitive signaling pathways. These features suggest dual structural and signaling roles for the dystrophin-glycoprotein complex. While much research has focused on protein-protein interactions that enable the DGC’s structural function, less is known about how the complex regulates signaling within muscle cells. Therefore, the goal of this thesis was to investigate the mechanisms whereby the dystrophin-glycoprotein complex regulates muscle nitric oxide (NO) production, a phenomenon that is crucial to normal muscle function and is disrupted in several forms of muscular dystrophy. A novel live cell imaging assay was developed to measure the mechanical activation of NO production in isolated muscle cells and investigate the biochemical signaling pathways involved in this process. This investigation identified dystrophin-dependent mechanoregulation of AMP-activated protein kinase (AMPK) as a key component of mechanosensitive NO production in striated muscle. Since defective muscle NO production contributes to diminished exercise tolerance in muscular dystrophy, subsequent studies investigated the therapeutic potential for acute pharmacologic AMPK activation to restore striated muscle NO production and improve exercise tolerance in a mouse model of dystrophin-deficient muscular dystrophy. Acute AMPK activation stimulated NO production in isolated dystrophin-deficient striated muscle cells in vitro, and increased the exercise capacity of dystrophin-deficient mice in vivo. These results suggest that acute AMPK activation may be a viable therapeutic strategy to improve exercise tolerance in muscular dystrophy patients. Finally, a novel transgenic mouse model was generated in order to test the contribution of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, to poor exercise tolerance in muscular dystrophy. These experiments suggested that ADMA contributes to exercise-induced fatigue in female dystrophin-heterozygous mice, a model for female carriers of Duchenne muscular dystrophy mutations. They also indicated that ADMA may affect exercise tolerance via effects to promote hypertrophy and impair the contractile function of the dystrophin-heterozygous heart. Considered together, the findings of this thesis support the idea that nitric oxide production is impaired in dystrophin-deficient muscle due to combined effects of disrupted intracellular signaling cascades and increased release of endogenous nitric oxide synthase inhibitors from damaged cells. Thus, this research provides evidence for the hypothesis that both the structural and signaling functions of the dystrophin-glycoprotein complex are critical for the appropriate regulation of striated muscle nitric oxide production.
Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates post-exercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically-necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac β-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers, or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in DMD patients.
Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder, characterized by progressive skeletal muscle wasting. The disease is caused by various types
of mutations in the dystrophin gene (DMD). The disease occurs at a frequency of
about 1 in 5000 male births, making it the most common severe neuro-muscular
disease. In addition to clinical examinations of muscle strength and function, diagnosis of DMD usually involves a combination of immunological assays using muscle
biopsies, typically immunohistochemistry and western blotting, and molecular
techniques such as DMD gene sequencing or Multiplex Ligation Dependent Probe
Amplification (MLPA) using blood samples. In fact, precise molecular diagnosis is a
prerequisite for determining the appropriate personalized therapeutic approach
such as exon-skipping, gene therapy or stem cell-based therapies in conjunction
with gene editing techniques like CRISPR-Cas9. However, the quest for reliable
biomarkers with high sensitivity and specificity for DMD from liquid biopsy is still
a hotspot of research, as such non-invasive biomarker(s) would not only facilitate
disease diagnosis but would also help in carrier detection, which will eventually
result in better disease management. In this chapter, we will illustrate the detailed
current and prospect strategies for disease.
New findings:
What is the central question of this study? We examined whether the macrophage-synthesized antioxidant 7,8-dihydroneopterin was elevated in Duchenne muscular dystrophy (DMD) patients. We then examined whether 7,8-dihydroneopterin could protect dystrophic skeletal mouse muscle from eccentric contraction-induced force loss and improve recovery. What is the main finding and its importance? Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients. 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss of dystrophic skeletal mouse muscle and accelerated recovery of force. These results suggest that eccentric contraction-induced force loss is mediated, in part, by an oxidative component and provides a potential protective role for 7,8-dihydroneopterin in DMD.
Abstract:
Macrophage infiltration is a hallmark of dystrophin-deficient muscle. We tested the hypothesis that Duchenne muscular dystrophy (DMD) patients would have elevated levels of the macrophage-synthesized pterins, neopterin and 7,8-dihydroneopterin, compared with unaffected age-matched control subjects. Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients, and 7,8-dihydroneopterin/creatinine was associated with patient age and ambulation. Urinary 7,8-dihydroneopterin corrected for specific gravity was also elevated in DMD patients. Given that 7,8-dihydroneopterin is an antioxidant, we then identified a potential role for 7,8-dihydroneopterin in disease pathology. We assessed whether 7,8-dihydroneopterin could: (i) protect against isometric force loss in wild-type skeletal muscle exposed to various pro-oxidants; and (ii) protect wild-type and mdx muscle from eccentric contraction-induced force loss, which has an oxidative component. Force loss was elicited in isolated extensor digitorum longus (EDL) muscles by 10 eccentric contractions, and recovery of force after the contractions was measured in the presence of exogenous 7,8-dihydroneopterin. 7,8-Dihydroneopterin attenuated isometric force loss by wild-type EDL muscles when challenged by H2 O2 and HOCl, but exacerbated force loss when challenged by SIN-1 (NO• , O2• , ONOO- ). 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss in mdx muscle. Isometric force production by EDL muscles of mdx mice also recovered to a greater degree after eccentric contractions in the presence of 7,8-dihydroneopterin. The results corroborate macrophage activation in DMD patients, provide a potential protective role for 7,8-dihydroneopterin in the susceptibility of dystrophic muscle to eccentric contractions and indicate that oxidative stress contributes to eccentric contraction-induced force loss in mdx skeletal muscle.
We tested the hypotheses that maintaining the activity of nitric oxide by L-arginine infusion would counteract the release of an endogenous nitric oxide synthase inhibitor, improve survival, and decrease intraoperative hypertension after infrarenal aortic cross-clamp surgery. Hindlimb ischemia was generated by infrarenal aortic cross-clamping and tying of the left femoral artery for 5 hours in rats with bilateral femoral and sciatic nerves cut. Mean blood pressure significantly increased during the 5-hour ischemic period in ischemic rats (no drug treatment). Baroreceptor function was inhibited in ischemic rats assessed by intravenous dose response to phenylephrine and nitroprusside after 5 hours of ischemia, suggesting baroreceptor resetting. In ischemic rats infused with L-arginine the intraoperative hypertension was prevented during the suggesting that this hypertension may be mediated by nitric oxide inhibition. The rates of survival and arrhythmias 2 hours after declamping were 50% in ischemic rats and 100% in ischemic rats treated with N-omega-nitro-L-arginine (a nitric oxide synthase inhibitor) 10 minutes before declamping. In ischemic rats infused with L-arginine the survival rate was significantly increased to 100% and the arrhythmic rate was inhibited. We conclude that L-arginine prevents hypertension during cross-clamping and decreases the mortality rate and arrhythmias after declamping by maintaining nitric oxide synthesis. These results suggest that humoral factors released from the ischemic hindlimb may inhibit endogenous nitric oxide production, thus contributing to intraoperative hypertension, arrhythmias, and high mortality rate after aortic cross-clamp surgery.
Numerous reports have indicated that the plasma concentration of endogenously produced inhibitors of nitric oxide synthase are elevated in human disease states. In this review we discuss recent advances in our understanding of the enzymes responsible for the synthesis of these inhibitors.
Asymmetric dimethylarginine (ADMA) is an endogenous methylated amino acid derived from arginine. ADMA can inhibit the activity of all isoforms of nitric oxide synthase (NOS). Protein arginine methyltransferases (PRMTs) catalyze the synthesis of ADMA and dimethylarginine dimethylaminohydrolase (DDAH) is responsible for the metabolism of this compound. ADMA enters cells through cationic amino-acid transporters (CATs), which are known to be y + carriers. Many factors can regulate the synthesis, transport, metabolism, or excretion of ADMA. In various pathological states such as hypercholesterolemia, hyperglycemia, hyperhomocysteinemia, hypertension, coronary artery disease, heart failure, and stroke, plasma levels of ADMA may increase two-or even tenfold, contributing to inhibition of NO synthesis and endothelial dysfunction. Impaired liver or renal function could also have an impact on the plasma concentration of ADMA. In some situations such as neurological disorders, decreased levels of ADMA are noted. It is very important to discover which states or drugs can increase or decrease the level of ADMA and what the mechanism of that action is (Adv Clin Exp Med 2010, 19, 233–243).
Oestrogenen en asymmetrische dimethylarginine Hart- en vaatziekten (HVZ) vormen de belangrijkste doodsoorzaak in de Westerse wereld. Premenopauzale vrouwen hebben een lager risico op HVZ dan mannen van dezelfde leeftijd, maar na de menopauze neemt de kans op HVZ bij vrouwen snel toe. Door de eierstokken geproduceerde hormonen (oestrogenen en progestagenen) spelen waarschijnlijk een rol bij de bescherming van premenopauzale vrouwen tegen HVZ en mogelijk kan toediening van deze hormonen na de menopauze vrouwen enigszins beschermen tegen de versnelde toename van het risico van HVZ. Asymmetrische dimethylarginine (ADMA) is een stof die door het lichaam zelf wordt gemaakt. Een hoge concentratie van ADMA vermindert een goed functioneren van de bloedvaten en leidt tot een verhoogde kans op HVZ. Of ADMA ook een rol speelt bij de verhoogde kans op HVZ na de menopauze is nog niet eerder onderzocht. Dit proefschrift behandeld het onderzoek naar de relatie tussen oestrogenen en aan oestrogeen verwante stoffen en ADMA bij vrouwen van middelbare leeftijd. Er werd gekeken naar de invloed van de menopauze en naar de effecten van verschillende (hormonale) menopauzale therapieën op ADMA concentraties.
De voornaamste bevinding was dat hogere oestrogeen concentraties gepaard gaan met lagere ADMA concentraties. Het ADMA verlagende effect van oestrogeensuppletie was groter bij orale toediening dan bij gebruik van pleisters of spray en bovendien
afhankelijk van de gebruikte oestrogeen/progestageen combinatie. Hoewel dit onderzoek laat zien dat vrouwelijke geslachtshormonen ADMA spiegels in gunstige zin beïnvloeden, moeten de klinische consequenties hiervan nog nader onderzocht worden.
Numerous reports have indicated that the plasma concentration of endogenously produced inhibitors of nitric oxide synthase
are elevated in human disease states. In this review we discuss recent advances in our understanding of the enzymes responsible
for the synthesis and metabolism of these inhibitors.
Recently described techniques for separating myosin isoenzymes have been adapted for analysis of myosins from diseased and developing human skeletal muscle. The method is highly suitable for analysis of human myosins because only 2–3 mg of muscle are required for routine analyses.Human embryonic/foetal myosins are electrophoretically distinct from mature skeletal myosins, and are not normally detected beyond the first month of post-natal life, except in premature infants. They have a high alkaline calcium-activated ATPase activity. This would account for the histochemical classification of foetal fibres as “Type II”, although physiological differences between adult fast-twitch muscle and foetal muscle are well recognized.Foetal myosins are also synthesized in human skeletal muscle under certain pathological circumstances. Their presence in Duchenne dystrophy probably reflects the associated marked muscle regeneration, with immaturity of some muscle cells. The large amounts of foetal myosin present in many cases of infantile spinal muscular atrophy is evidence that innervation is necessary for the normal cessation of foetal myosin synthesis.
Dimethylarginine dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine (ADMA), an endogenously produced nitric oxide (NO) synthase inhibitor. In mammals, two isoforms of DDAH, DDAH1 and DDAH2, are expressed in the cardiovascular system, suggesting that ADMA concentrations are actively regulated in blood vessels, raising the possibility that cardiovascular metabolism of ADMA constitutes a novel mechanism for the regulation of NO production. The purpose of this study was to determine the role of DDAH-catalyzed asymmetric methylarginine metabolism in the regulation of vascular function. We developed adenoviral vectors for the expression of human DDAH1 and 2. Overexpression of DDAH1 or 2 in human umbilical vein endothelial cells (HUVEC) increases DDAH activity, reduces ADMA concentrations and increases NO production. Similarly, overexpression of DDAH1 or 2 in DDAH1(+/-) mice carotid vessels increases NO production and attenuates the response to phenylephrine (PE), enhances acetylcholine (ACh) relaxation and attenuates the effect of exogenously applied ADMA. Finally, overexpression of either DDAH1 or 2 completely reversed the vascular dysfunction seen in DDAH1(+/-) mice. These data indicate that basal concentrations of ADMA in blood vessels are sufficient to regulate NO production, that increases in the level of either DDAH1 or 2, improves vascular function and that overexpression of either DDAH1 or 2 is sufficient to compensate for life-long exposure to elevated ADMA. Thus, therapeutic manipulation of DDAH expression or activity may represent a novel approach to improve vascular dysfunction in various cardiovascular diseases.
The metabolic fates of NG,NG-and NG,N'G-dimethylarginines in rats were investigated isotopically and novel metabolites, alpha-keto-delta-(N,N-dimethylguanidino)-and alpha-keto-delta-(N,N'-dimethylguanidino)valeric acids and gamma-(N,N-dimethylguanidino)-and gamma-(N,N'-dimethylguanidino)butyric acids were identified. In the case of the rats injected with NG,NG-dimethyl-L-[1,2,3,4,5-14C]arginine, about 13% of the radioactivity was recovered in the first 12-h urine and was distributed in the following metabolites (relative ratios): unchanged NG,NG-dimethyl-L-arginine (35.2%), gamma-(N,N-dimethylguanidino)butyric acid (18.4%), alpha-keto-delta-(N,N-dimethylguanidino)valeric acid (16.4%), and N alpha-acetyl-NG,NG-dimethyl-L-arginine (8.5%). The radioactivity retained in the tissues was found mainly in citrulline and was further distributed in ornithine, arginine, and glutamic acid and even in protein-bound arginine. In the case of NG,N'G-dimethyl-L-[1,2,3,4,5-14C]arginine-injected rats, about 75% of the radioactivity was excreted in the first 12-h urine and was recovered in the following metabolites (relative ratios): N alpha-acetyl-NG,N'G-dimethyl-L-arginine (48.4%), unchanged NG,N'G-dimethyl-L-arginine (23.7%), alpha-keto-delta-(N,N'-dimethylguanidino)valeric acid (20.2%), and gamma-(N,N'-dimethylguanidino)butyric acid (9.6%). In the tissues, most of the radioactivity was associated with unchanged NG,N'G-dimethyl-L-arginine. These findings show that both dimethylarginines are metabolized by a pathway forming the corresponding alpha-ketoacid analogs and the oxidatively decarboxylated products of the alpha-ketoacids in addition to the N alpha-acetyl conjugates identified previously (K. Sasaoka, T. Ogawa, and M. Kimoto (1982) Arch. Biochem. Biophys. 219, 454-458), and NG,NG-dimethyl-L-arginine is catabolized by an additional pathway leading to the formation of citrulline and its metabolically related amino acids. By considering their catabolism, an attempt to use urinary dimethylarginines as an index of in vivo breakdown of tissue proteins is invalid at least in rats.
The excretion of 3-methylhistidine increased in the urine of dystrophic mice C57BL/6J. The content of 3-methylhistidine residue decreased in the muscle proteins of dystrophic mice, but not in other organs. Methylated proteins in the skeletal muscle, actin and myosin, were partially purified from the dystrophic and control muscles. The amount of 3-methylhistidine residue in unit weight of the actin and myosin preparations was normal in dystrophic muscle. These three facts indicate that the turnover rates of actin and myosin are increased in the muscle of the dystrophic mice.
Urinary excretion of the post-translationally modified amino-acid 3-methylhistidine, derived from the contractile proteins actin and myosin, was measured in patients with conditions associated with nitrogen loss. The ratio of 3-methylhistidine:creatinine excretion, a measure of the fractional catabolic rate of myofibrillar protein was increased in severe injury, thyrotoxicosis, neoplastic disease, prednisolone administration, and sometimes Duchenne muscular dystrophy. In myxoedema, osteomalacia, and hypothermia the ratio was decreased; and starvation, elective operations, and rheumatoid arthritis had little effect. Provided that the diet is meat free, measurement of urinary 3-methylhistidine may provide useful information on the cause of protein loss.
N tau-Methylhistidine (MH) and creatinine levels were determined in amniotic fluid and maternal serum from 81 women undergoing midtrimester amniocentesis for reasons other than the diagnosis of neuromuscular disease. Samples were also examined in three pregnancies with male fetuses who were subsequently found to have Duchenne muscular dystrophy (DMD). Between 16 and 20 weeks' gestation, amniotic fluid and maternal serum MH levels averaged 3.22 and 1.94 mumoles/L, respectively. No significant differences were found between the control and affected fetuses for MH and creatinine levels or for MH/creatinine ratios from amniotic fluid or maternal serum. Determination of amniotic fluid MH level thus has no apparent value in the prenatal diagnosis of DMD.
We tested the hypotheses that maintaining the activity of nitric oxide by L-arginine infusion would counteract the release of an endogenous nitric oxide synthase inhibitor, improve survival, and decrease intraoperative hypertension after infrarenal aortic cross-clamp surgery. Hindlimb ischemia was generated by infrarenal aortic cross-clamping and tying of the left femoral artery for 5 hours in rats with bilateral femoral and sciatic nerves cut. Mean blood pressure significantly increased during the 5-hour ischemic period in ischemic rats (no drug treatment). Baroreceptor function was inhibited in ischemic rats assessed by intravenous dose response to phenylephrine and nitroprusside after 5 hours of ischemia, suggesting baroreceptor resetting. In ischemic rats infused with L-arginine the intraoperative hypertension was prevented during the 5-hour period, suggesting that this hypertension may be mediated by nitric oxide inhibition. The rates of survival and arrhythmias 2 hours after declamping were 50% in ischemic rats and 100% in ischemic rats treated with N omega-nitro-L-arginine (a nitric oxide synthase inhibitor) 10 minutes before declamping. In ischemic rats infused with L-arginine the survival rate was significantly increased to 100% and the arrhythmic rate was inhibited. We conclude that L-arginine prevents hypertension during cross-clamping and decreases the mortality rate and arrhythmias after declamping by maintaining nitric oxide synthesis. These results suggest that humoral factors released from the ischemic hindlimb may inhibit endogenous nitric oxide production, thus contributing to intraoperative hypertension, arrhythmias, and high mortality rate after aortic cross-clamp surgery.
We tested the hypothesis that the endogenous nitric oxide synthetase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), regulates cardiovascular function by central mechanisms. In in vivo studies, rats received intracerebroventricular (i.c.v.) injection of isotonic saline, ADMA (1 mg), l-arginine (3 mg), and N omega-nitro-l-arginine methylester (l-NAME, 1 mg). Baroreflex function was then assessed by intravenous (i.v.) injection of phenylephrine. Central application of exogenous NOS inhibitor, l-NAME, increased mean arterial blood pressure and decreased heart rate. However, application of the endogenous NOS inhibitor, ADMA, decreased mean arterial blood pressure and heart rate simultaneously (-39 +/- 6 mm Hg and -50 +/- 8 beats/min, respectively). Both l-NAME (i.c.v.) and ADMA (i.c.v.) significantly inhibited the baroreflex function, indicating a regulatory role of central nitric oxide in controlling baroreflex function. In contrast to the central effect, intravenous injection of ADMA caused dose-dependent increases in mean arterial blood pressure that could be blocked by l-NAME pretreatment. In vitro studies using aortic rings demonstrated that ADMA (10(-4)M) significantly increased the concentration of acetylcholine for the threshold response (EC15) and half-maximal response (EC50). This indicates that ADMA inhibits the constitutive isoform of NOS in the endothelium. ADMA may have functional importance in regulating cardiovascular function by mechanisms in addition to the inhibition of nitric oxide synthesis.
An increasing number of reports in the literature indicate that endogenously produced inhibitors of nitric oxide synthase (NOS), particularly asymmetric dimethylarginine (ADMA) regulate nitric oxide generation in numerous disease states. Two dimethylarginine dimethylaminohydrolase (DDAH) enzymes metabolise ADMA. We and others have postulated that activity of DDAH is a key determinant of ADMA levels in vivo. This review summarises recent advances in the regulation and function of DDAH enzymes and its role in the regulation of nitric oxide generation.
Numerous reports have indicated that the plasma concentration of endogenously produced inhibitors of nitric oxide synthase are elevated in human disease states. In this review we discuss recent advances in our understanding of the enzymes responsible for the synthesis of these inhibitors.
NO beating. Nω,Nω-Dimethyl-L-arginine dimethylaminohydrolase (DDAH, see Figure) is a regulator of NO production in mammalian organisms. Among the many approaches to inhibit NO production for the treatment of severe diseases like migraine, ischemia, septic shock, or cancer, inhibition of DDAH is a novel and promising one. Research progress in its (patho)physiology and biochemistry is described and discussed in this article.
The metabolic status of 15 intensive care patients receiving a standardized total parenteral nutrition regimen was followed up to 15 days immediately after admission by measuring 3-methylhistidine, total nitrogen, and creatinine excretion. The average 3-methylhistidine excretion was within the normal range during the first 3 days, rising on day 4 and reached a maximum of 70% above normal values on day 5. It declined to within normal range thereafter in most of the patients. Mean values for creatinine excretion remained relatively constant within the normal range throughout the study. During all days 3-methylhistidine was negatively correlated with N-balance. It is concluded that these patients had increasing catabolism with a maximum on day 5 and that the catabolic condition was associated with an increased muscle protein breakdown.
1. Eight different substances were isolated in crystalline forms by ion exchange chromatography of the aliphatic basic amino acid fraction of human urine.
2. Structures of guanidino-N,N-dimethylarginine and guanidino-N,N'-dimethylarginine were assigned for two substances by analyses of the degradation products, elemental compositions, and nuclear magnetic resonance spectra. The identities of the compounds were confirmed by synthesis followed by comparison of infrared and nuclear magnetic resonance spectra and of chromatographic properties with those of the isolated compounds. The concentrations of these two compounds in human urine are higher than that of arginine. Their natural occurrence has not been reported previously.
3. Three compounds, Nε-methyl-, Nε,Nε-dimethyl-, and Nε,Nε,Nε-trimethyllysine, were identified by elementary analyses and syntheses. Occurrence of the last two compounds in free state in nature has not been reported previously.
4. Two compounds, glucosylgalactosyl- and galactosyl-δ-hydroxylysine, whose occurrence in human urine has been reported previously, were isolated as crystals for the first time.
5. The concentrations of these N-methyl derivatives of lysine and arginine in the urine were not changed by oral loading of either lysine or arginine. Protein-free diet did not affect the amounts of these compounds in urine. The possibility that these compounds are derived from tissue proteins in which lysine and arginine residues are methylated was discussed.
NG, () has now been found to be present in myosin prepared from developing leg muscle. Neither monomethylarginine nor (NG) could be detected in our preparations. Cultured muscle cell myosin contains up to four residues of this amino acid per 5 × 105 grams of protein. Myosins from leg muscle of chick embryos and neonatal rats contained less than two residues of , while none was detected in adult chicken or rat myosins. Cardiac myosins from developing as well as mature animals lacked or contained very little . Actin was devoid of this amino acid.
The newly available spherical cation-exchange resins HP-AN 90 and HP-B 80 are capable of performing various analyses of amino acids and peptides at low backpressures. These multipurpose resins can perform: a 2 or 4 hr simple (approximately 20 amino acids) procedure used with protein hydrolyzates, or a complex (50 or more amino acids) technical procedure (for physiological fluids), or a rapid screening procedure dealing with specific amino acids as they relate to clinical disease or biomedical studies, or the analysis of peptide samples.Analysis time for acidic and neutral amino acids in a physiological fluid, using sodium citrate buffers, has been reduced from the previously reported 11.5 hr to about 6 hr. With lithium citrate buffers, the chromatographic separation of glutamine and asparagine without sacrifice of resolution of other amino acids can now be completed in 205 min using the same HP-AN 90 resin. It is now possible to analyze four complex samples (acidic, neutral, and basic components) per day.
—Methods for the determination of methyl-lysine, methyllarginine and methylhistidine residues of tissue proteins are described. They consist of preliminary purification of basic amino acids, enzymic removal of lysine, arginine and histidine followed by amino acid analysis. Recovery rates and specificities of the method were satisfactory. The contents of methylamino acids in proteins of mammalian organs were determined.
The distribution of proteins containing the methylamino acids in human brain showed that the concentrations of methyl-lysine and NG,N′G-dimethylarginine were highest in the gray matter of the cerebellar cortex and relatively high in regions rich in gray matter, while those of NG-mono- and NG,N′G-dimethylarginine were highest in the white matter. The following findings suggest that most of the NG-mono- and NG,N′G-dimethylarginine was associated with the myelin basic protein. The distribution of the methylarginine residues of acid-soluble proteins in bovine brains coincided with the cerebroside pattern. The concentrations of the amino acids in acid-soluble proteins of rat brain increased concomitantly with the increase of cerebroside. The methylamino acid content in proteins increased during the purification of the myelin basic protein from the white matter of human and bovine brains.
Proteins containing NG,NG-dimethyiarginine and di- and trimethyl-lysine are concentrated in cell nuclei. The first amino acid was found mainly in nucleoplasmic proteins and the other two were found in histones. The concentration of 3-methylhistidine residue, highest in muscular proteins, is low in cerebral proteins and is probably derived from proteins of walls of blood vessels in the brain.
Myofibrillar protein catabolic rate was calculated in seven patients with Duchenne muscular dystrophy from the amount of 3-methylhistidine excreted in the urine, and found to be over three times that found in a control series when expresses as the percentage of myofibrillar protein catabolised per day. It is suggested that measurement of myofibrillar protein catabolic rate may add a useful parameter in the study of muscle disorders.
Samples of psoas muscle from nine infants (aged 1 day to 14 mo) and of several skeletal muscles from seven adult males (age 19-74 yr) were analyzed for content of protein-bound Ntau-methylhistidine (3-methylhistidine; 3-Mehis). The mean content of 3-Mehis (expressed as mumoles/g mixed protein) was 3.2 (range 2.4-3.7) in infants and 4.2 (range 3.7-4.6) in adults. The daily urinary excretion of 3-Mehis was measured in four young adult males receiving an egg-protein, flesh-free diet. Mean excretion of 3-Mehis was 211 (range 167-252) mumoles/day. From these two sets of data the mean rate of muscle protein breakdown in adult males was estimated to be 50 g/day, or 0.7 +/- 0.1 g/kg body weight/day. These results are compared with reported values for the 3-Mehis content of mixed proteins in muscle of various species, and with published estimates, computed by other techniques, of the rate of muscle protein breakdown in human subjects.
Nuclear proteins have been fractionated into five distinct classes according to their extractability from rat liver nuclei at different pH and salt concentrations. The fractions have been analyzed for their amino acid composition which shows the presence of NG, NG-dimethylarginine, in sizable amount, in non-histone nuclear proteins (NHNP). This modification is most prominent in proteins which are found associated with rapidly-labeled heterogeneous RNA (HnRNP proteins).
L-(Methyl-14C)-methionine was administered i.p. to mice, and the incorporation of radioactive methionine into proteins and methyllysine and methylarginine residues formed by the transfer of the methyl-14C group of methionine were measured. Tissue protein was actively methylated in organs having a high activity of protein synthesis, and the in vivo methylating activity in organs was not correlated with theprotein methylating activity of the organs determined in vitro. Puromycin inhibited both protein synthesis and protein methylation in mouse organs to a similar degree. Neither the formation of S-adenosyl-(methyl-14C)-methionine nor protein methylase was inhibited by puromycin. The data suggests that proteins are methylated immediately after protein synthesis, that is, newly synthesized proteins are the substrates of protein methylation. Radioactive methionine and the [C14] methyl groups of methyllysine and methylarginine residues of tissue proteins are degraded in parallel over a period of 3 wk, suggesting that protein methylation is an irreversible type of protein modification.
In order to use Ntau-methylhistidine (3-methylhistidine) excretion in the urine as a measure of muscle protein breakdown, it is necessary to demonstrate that other tissues are not important sources of this protein constituent. Accordingly, the concentration of Ntau-methylhistidine in blood serum and in the mixed proteins of heart, brain, lung, kidney, diaphragm, spleen, testis, stomach, liver and hind leg skeletal muscle was measured in male rats of approx. 400 g body weight. The free Ntau-methylhistidine concentration of rat serum was less than 2 nmol per ml. In contrast, measurable amounts of Ntau-methylhistidine were found in the mixed proteins of all tissues and organs examined. The highest concentration was found in skeletal muscle (658 nmol/g tissue). Assuming muscle mass to be 45% of body weight, it has been estimated that the muscle contains more than ten times the total amount of this amino acid present in all of the other organs analyzed, which together account for about 20% of total body weight. These findings indicate that skeletal muscle is likely to be the major source of urinary Ntau-methylhistidine and the latter is, in consequence, a reflection of myofibrillar protein breakdown in skeletal muscle.
Tallan, Stein, and Moore (1954) identified 3-methylhistidine (3-MeHis) as a component of human urine, and commented that the source of this material was far from clear. The results of analyses made in this laboratory on samples of blood and urine collected under fasting conditions indicated that urinary 3-MeHis has an endogenous origin. Because of its possible physiological significance attempts were made to locate its source. Evidence will be presented here that 3-MeHis is a component of actin, an important functional protein of muscle.