[The evaluation of homoarginine and folic acid in patients with arterial hypertension.] / Оценка гомоаргинина и фолиевой кислоты у пациентов с артериальной гипертензией.

According to current data, a low level of folic acid (FA) contributes to the progression of arterial hypertension (AH), affecting the metabolism of cells that are involved in the vascular tone regulation, such as hypothalamic astrocytes of nervous tissue. It is also known that the level of FA in the nervous tissue and cerebrospinal fluid is 2-3 times higher than in plasma. There is another metabolic marker of cardiovascular diseases, the level of plasma homoarginine (hArg). The decrease in the level of plasma hArg is also known as a diagnostic sign. In our study, we established that in patients with AH (n = 60), the level of hArg was almost 2 times lower than in healthy individuals, and in 75% of cases the rate was below 1.80 µM. The insufficiency of FA taking into account its low level in plasma FA, as well as the level of total homocysteine (tHcy) higher than 10.9 µM, was observed in 78% of patients. HArg levels at values less than 1.80 µM corresponded to a statistically significant decrease in FA when its content was less than 13.5 nM. This relationship (r = 0.63, p = 0.020) appears in patients with AH, regardless of the number and severity of target organ damage (TOD). FA and hArg as metabolic markers exhibit various diagnostic capabilities when comparing subgroups of patients without TOD and with multiple TOD. Significant differences fared at an acceptable level (p = 0.007) only for the hArg levels, while for the FA concentrations there was only a trend to decrease. It is possible that metabolic disturbances in the central nervous system that are associated with the necessary to maintain high FA concentration contribute to the development of hypertensive status. The causal relationship of a parallel decrease in hArg and FA levels in patients with AH requires further research.

Synthesis of homoagmatine and GC-MS analysis of tissue homoagmatine and agmatine: evidence that homoagmatine but not agmatine is a metabolite of pharmacological L-homoarginine in the anesthetized rat.

Low L-homoarginine (hArg) concentrations in human blood and urine are associated with renal and cardiovascular morbidity and mortality, yet the underlying mechanisms and the biological activities of hArg are elusive. In humans and rats, hArg is metabolized to L-lysine. The aim of the present work was to study hArg metabolism to agmatine (Agm) and homoagmatine (hAgm) in the anesthetized rat. Using a newly developed and validated GC-MS method and a newly synthesized and structurally characterized hAgm we investigated the metabolism of i.p. administered hArg (0, 20, 220, 440 mg/kg) to hAgm and Agm in lung, kidney, liver and heart in anesthetized rats. Our study provides unequivocal evidence that hArg is metabolized to hAgm but not to Agm. Whether hAgm derived from hArg's metabolism may contribute to the pathophysiological significance of endogenous hArg and for the favoured effects of pharmacological hArg remains to be demonstrated. The biology of hArg warrants further investigations.

Utility of homoarginine in the GC-MS quantification of asymmetric dimethylarginine (ADMA) in human serum proteins.

NG,NG-Dimethyl-L-arginine (asymmetric dimethylarginine, ADMA) is a cardiovascular risk factor. ADMA circulates in blood as a free acid (fADMA) and as constituent of not yet identified proteins (prADMA). We describe here a protocol for the GC-MS quantification of ADMA released from serum proteins using 6 M HCl (110 °C, 20 h). L-Homoarginine (hArg) is useful in measuring digestibility of amino acids in food proteins. We demonstrate that hArg is not present in human serum proteins and is useful in measuring serum prADMA. The concentration of prADMA in elderly subjects is about 90 nM and the average fADMA/prADMA ratio 6:1.

Plasma Homoarginine Concentrations According to Use of Hormonal Contraception.

Estrogen is a potent vasodilator through activation of endothelial nitric oxide synthase (eNOS). Arginine and its homologue homoarginine are substrates for NOS, while asymmetric dimethylarginine (ADMA) is a NOS inhibitor. Healthy, never-pregnant women aged 18 to 40 years (n = 158) were categorized according to use of hormonal contraception into non-users (n = 76), users of estrogen contraceptives (EC-users, n = 58) and users of progestins-only contraceptives (PC-users, n = 24). Plasma homoarginine, arginine, ADMA and SDMA concentrations were assayed using a LC-MS/MS method. Compared to non-users, EC users had higher plasma homoarginine (median (interquartile range) 1.63 (1.24, 2.04) vs. 2.39 (2.05, 2.85) µmol/L, p < 0.001), lower arginine (80.8 (72.4, 94.3) vs. 72.1 (62.9, 85.1) µmol/L, p = 0.008) and ADMA (0.52 (0.46, 0.59) vs. 0.48 (0.42, 0.54) µmol/L, p = 0.003) concentrations. The lowest median plasma homoarginine concentration (1.34 (0.92, 1.75) µmol) was seen in PC-users. No differences were seen in SDMA concentrations according to use of hormonal contraception. In healthy, never-pregnant women aged 18 to 40 years, use of estrogen containing contraception was associated with significantly higher plasma concentrations of homoarginine and lower plasma concentrations of arginine and ADMA as compared to non-users, while the lowest plasma homoarginine concentrations were seen in progestin-only users. Whether the observed changes in relation to use of hormonal contraception have an impact on cardiovascular status, should be evaluated in an intervention study.

Markers of nitric oxide are associated with sepsis severity: an observational study.

BACKGROUND: Nitric oxide (NO) regulates processes involved in sepsis progression, including vascular function and pathogen defense. Direct NO measurement in patients is unfeasible because of its short half-life. Surrogate markers for NO bioavailability are substrates of NO generating synthase (NOS): L-arginine (lArg) and homoarginine (hArg) together with the inhibitory competitive substrate asymmetric dimethylarginine (ADMA). In immune cells ADMA is cleaved by dimethylarginine-dimethylaminohydrolase-2 (DDAH2). The aim of this study was to investigate whether concentrations of surrogate markers for NO bioavailability are associated with sepsis severity. METHOD: This single-center, prospective study involved 25 controls and 100 patients with surgical trauma (n = 20), sepsis (n = 63), or septic shock (n = 17) according to the Sepsis-3 definition. Plasma lArg, hArg, and ADMA concentrations were measured by mass spectrometry and peripheral blood mononuclear cells (PBMCs) were analyzed for DDAH2 expression. RESULTS: lArg concentrations did not differ between groups. Median (IQR) hArg concentrations were significantly lower in patient groups than controls, being 1.89 (1.30-2.29) µmol/L (P < 0.01), with the greatest difference in the septic shock group, being 0.74 (0.36-1.44) µmol/L. In contrast median ADMA concentrations were significantly higher in patient groups compared to controls, being 0.57 (0.46-0.65) µmol/L (P < 0.01), with the highest levels in the septic shock group, being 0.89 (0.56-1.39) µmol/L. The ratio of hArg:ADMA was inversely correlated with disease severity as determined by the Sequential Organ Failure Assessment (SOFA) score. Receiver-operating characteristic analysis for the presence or absence of septic shock revealed equally high sensitivity and specificity for the hArg:ADMA ratio compared to the SOFA score. DDAH2 expression was lower in patients than controls and lowest in the subgroup of patients with increasing SOFA. CONCLUSIONS: In patients with sepsis, plasma hArg concentrations are decreased and ADMA concentrations are increased. Both metabolites affect NO metabolism and our findings suggest reduced NO bioavailability in sepsis. In addition, reduced expression of DDAH2 in immune cells was observed and may not only contribute to blunted NO signaling but also to subsequent impaired pathogen defense.

Determination of ß-N-oxalyl-L-α,ß-diaminopropionic acid and homoarginine in Lathyrus sativus and Lathyrus cicera by capillary zone electrophoresis.

BACKGROUND: Lathyrus species as legumes represent an alternative protein source for human and animal nutrition. Heavy consumption of these species can lead to lathyrism, caused by the non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP). Currently, there is no well-defined level below which ß-ODAP is considered non-toxic. In this work, the ß-ODAP content was determined in L. sativus and L. cicera samples to assess their potential toxicity. Homoarginine is another non-protein amino acid found in Lathyrus spp. with interesting implications for human and animal nutrition. RESULTS: The level of ß-ODAP found in these two species ranged from 0.79 to 5.05 mg g(-1). The homoarginine content of the samples ranged from 7.49 to 12.44 mg g(-1). CONCLUSION: This paper describes an accurate, fast and sensitive method of simultaneous detection and quantification of ß-ODAP and homoarginine by capillary zone electrophoresis in L. cicera and L. sativus seeds. Moreover, several methods of extraction were compared to determine the highest performance.

Homoarginine, ß-ODAP, and asparagine contents of grass pea landraces cultivated in Turkey.

The seeds of grass pea (Lathyrus sativus L.), a drought tolerant crop, were analysed for quantitative determination of the free amino acids ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), homoarginine and asparagine by a simple and fast capillary electrophoretic method. In boric acid (80mM, pH 8.0) running buffer system, not only were α and ß-ODAP successfully separated, but also an efficient sample stacking was achieved during hydrodynamic sample introduction. The validated method was used for quantification of ß-ODAP, homoarginine and asparagine in seed extracts of 52 Lathyrus local landraces from various regions of Turkey and one released cultivar. The concentration ranges of amino acids were found as 0.21-1.27% (w/w) for homoarginine, 0.10-0.87% (w/w) for ß-ODAP and 0.006-0.47% (w/w) for asparagine. A positive correlation between homoarginine and ß-ODAP quantities in seeds of 53 Lathyrus local landraces was shown to exist (r(2)=0.649).

A look at the brighter facets of ß-N-oxalyl-l-α,ß-diaminopropionic acid, homoarginine and the grass pea.

The low incidence of neurolathyrism, its absence in several communities traditionally consuming Lathyrus sativus and the likely benefits of its inclusion as part of a normal diet are reviewed. The metabolism/detoxification of ODAP which is unique to humans may be a crucial factor in this regard. The presence of homoarginine in the pulse which has received scant attention in the past could make this an invaluable pulse since it could contribute to a sustained generation of NO. NO is well recognized for its role in cardiovascular physiology and general well-being and thus a daily dietary intake of homoarginine through small quantities of L. sativus may be of advantage and deserves to be exploited. The detoxification of ODAP in humans could spotlight the pulse further for its non-neurotoxic attributes. Activation of PKC by ODAP adds a new dimension to explore its possible therapeutic potentials in areas such as Alzheimer's disease, hypoxia, and long term potentiation. These novel approaches to both ODAP and homoarginine might entirely change our perception of this poor man's pulse.

Evaluation of the furosine and homoarginine methods for determining reactive lysine in rumen-undegraded protein.

Three samples of soybean meal (SBM), 3 samples of expeller SBM (SoyPlus, West Central Cooperative, Ralston, IA), 5 samples of distillers dried grains with solubles (DDGS), and 5 samples of fish meal were used to evaluate the furosine and homoarginine procedures to estimate reactive Lys in the rumen-undegraded protein fraction (RUP-Lys). One sample each of SBM, expeller SBM, and DDGS were subjected to additional heat treatment in the lab to ensure there was a wide range in reactive RUP-Lys content among the samples. Furosine is a secondary product of the initial stages of the Maillard reaction and can be used to calculate blocked Lys. Homoarginine is formed via the reaction of reactive Lys with O-methylisourea and can be used to calculate the concentration of reactive Lys. In previous experiments, each sample was ruminally incubated in situ for 16 h, and standardized RUP-Lys digestibility of the samples was determined in cecectomized roosters. All rumen-undegraded residue (RUR) samples were analyzed for furosine and Lys; however, only 9 of the 16 samples contained furosine, and only the 4 unheated DDGS samples contained appreciable amounts of furosine. Blocked RUP-Lys was calculated from the furosine and Lys concentrations of the RUR. Both the intact feed and RUR samples were evaluated using the homoarginine method. All samples were incubated with an O-methylisourea/BaOH solution for 72 h and analyzed for Lys and homoarginine concentrations. Reactive Lys concentrations of the intact feeds and RUR were calculated. Results of the experiment indicate that blocked RUP-Lys determined via the furosine method was negatively correlated with standardized RUP-Lys digestibility, and reactive RUP-Lys determined via the guanidination method was positively correlated with standardized RUP-Lys digestibility. Reactive Lys concentrations of the intact samples were also highly correlated with RUP-Lys digestibility. In conclusion, the furosine assay is useful in predicting RUP-Lys digestibility of DDGS samples, and the guanidination procedure can be used to predict RUP-Lys digestibility of SBM, expeller SBM, DDGS, and fish meal samples.

Chemical modulation of the chaperone function of human alphaA-crystallin.

alphaA-crystallin is abundant in the lens of the eye and acts as a molecular chaperone by preventing aggregation of denaturing proteins. We previously found that chemical modification of the guanidino group of selected arginine residues by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), makes human alphaA-crystallin a better chaperone. Here, we examined how the introduction of additional guanidino groups and modification by MGO influence the structure and chaperone function of alphaA-crystallin. alphaA-crystallin lysine residues were converted to homoarginine by guanidination with o-methylisourea (OMIU) and then modified with MGO. LC-ESI-mass spectrometry identified homoargpyrimidine and homohydroimidazolone adducts after OMIU and MGO treatment. Treatment with 0.25 M OMIU abolished most of the chaperone function. However, subsequent treatment with 1.0 mM MGO not only restored the chaperone function but increased it by approximately 40% and approximately 60% beyond that of unmodified alphaA-crystallin, as measured with citrate synthase and insulin aggregation assays, respectively. OMIU treatment reduced the surface hydrophobicity but after MGO treatment, it was approximately 39% higher than control. FRET analysis revealed that alphaA-crystallin subunit exchange rate was markedly retarded by OMIU modification, but was enhanced after MGO modification. These results indicate a pattern of loss and gain of chaperone function within the same protein that is associated with introduction of guanidino groups and their neutralization. These findings support our hypothesis that positively charged guanidino group on arginine residues keeps the chaperone function of alphaA-crystallin in check and that a metabolic alpha-dicarbonyl compound neutralizes this charge to restore and enhance chaperone function.

Long-term effect of partial nephrectomy on biological parameters, kidney histology, and guanidino compound levels in mice.

The long-term adverse consequences of early renal mass reduction in mice have not yet been investigated. The effects of partial surgical nephrectomy (NX) in 2-month-old mice on some biological parameters, on histopathologic and morphometric features of the kidney, and on urea and guanidino compound (GC) levels in plasma, urine, and brain were examined at 10 days, and 1, 2, 4, and 12 months postsurgery. Body weight, urinary volume, and plasma urea were most affected at 10 days and 12 months post-NX. NX-induced changes in the remaining renal tissue (including hypertrophy, glomerular mesangial expansion, and presence of protein casts) increased with age. As in human renal insufficiency, NX mice showed significantly higher plasma guanidinosuccinic acid (GSA) and creatinine (CTN) levels at all studied periods. The same tendency could be seen for most other plasma GCs examined, except for arginine (Arg), guanidinoacetic acid (GAA), and homoarginine (HA). As seen in human pathobiochemistry, the latter 2 compounds tended to be lower in NX mice in our follow-up study. Remarkably, and also similar to humans, NX mice excreted less GAA and more GSA than controls during the entire follow-up study. During the follow-up, excretion levels of GAA were unchanged in NX and sham-operated mice. In brain, GAA and gamma-guanidinobutyric acid (GBA) levels were always higher in NX mice with a tendency to respectively increase or decrease over time in NX as well as sham-operated mice. Although urea and GC metabolism were influenced by time post-NX and aging, the model was confirmed to display a mild stable chronic impairment of renal function. Histopathologic and morphometric changes of the kidney increased with age.

Analysis of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid and homoarginine in Lathyrus sativus by capillary zone electrophoresis.

A simple capillary zone electrophoresis (CZE) method has been developed for the simultaneous quantitative determination of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP) and homoarginine in Lathyrus sativus (LS; grass pea). A new Na2B4O7-Na2SO4 run buffer was used and the pH was 9.20, contents of beta-ODAP and homoarginine in crude extracts of LS plant material were determined with this method, the RSDs of peak areas of beta-ODAP and homoarginine were 2.62% and 3.61%, respectively. It was found that the equilibrium concentration ratio of alpha- and beta-ODAP decreased from 34.5/65.5 to 28.6/71.4 when the pH of the solution increased from pH 3.0 to pH 11.0.

Secretion of homoarginine into the gut of chickens.

A technique, based on the homoarginine present in guanidinated proteins, has been used to distinguish between endogenous secretions and exogenous dietary amino acids in the ileal digesta of monogastric animals. This technique assumes that the ingested homoarginine is not recycled into the small intestine after absorption, but this assumption is yet to be experimentally validated in chickens. The secretion of homoarginine into the gut of broilers that were intravenously infused with 20 and 40 mmol/L homoarginine solutions was assessed. The plasma concentrations of homoarginine increased with increasing concentrations of homoarginine infused. However, only negligible levels of homoarginine (7.0 to 45.2 micrograms/g dry matter) were found in the digesta. Less than 0.01% of the intravenously infused homoarginine was recovered in the intestinal digesta, indicating that the secretion of homoarginine into the gut of chickens was insignificant.

Evaluation of homoarginine as a marker for the determination of endogenous amino acid concentrations in poultry excreta.

1. Endogenous amino acid losses in ileal digesta and excreta of adults cockerels fed on diets containing guanidinated forms of casein, soyabean meal and cottonseed meal were determined using homoarginine as a marker. 2. The ileal endogenous amino acid losses were markedly higher (P < 0.001) in birds given the cottonseed meal diet compared to those given the other two diets. The ileal endogenous protein was rich in aspartic acid, serine and glutamic acid. 3. Negative values were obtained for endogenous amino acid output in excreta. These aberrant values were caused by high concentrations of homoarginine in the excreta. A subsequent study with broiler chickens showed that the homoarginine in excreta was of urinary origin. 4. These results indicate that the homoarginine technique is not suitable for determining endogenous amino acid losses in excreta, but applicable when determinations are made in the terminal ileum.

Soybean trypsin inhibitor(s) reduce absorption of exogenous and increase loss of endogenous protein in miniature pigs.

It was the purpose of this study to define whether trypsin inhibitors impair protein digestibility via enhanced loss of exogenous or endogenous protein by quantifying those losses using the homoarginine technique, recently developed in this laboratory. Pigs fitted with permanent ileal T-cannulas were fed test meals containing homoarginine-labeled protein. The meals contained casein and increasing doses of trypsin inhibitors (Experiment 1) or alternatively either heat-treated or raw ground soybeans (Experiment 2). Following a casein meal (425 mmol nitrogen, no trypsin inhibitors), ileal protein was predominantly of endogenous rather than of exogenous origin (105 vs. 9 mmol nitrogen). Addition of isolated trypsin inhibitors (3000 mg) enhanced appearance of both endogenous and exogenous protein at the ileum (by 73 and 9 mmol nitrogen, respectively). Feeding raw instead of heat-treated soybeans in one single test meal caused a significant increase of endogenous protein from 217 +/- 42 to 263 +/- 47 mmol (mean +/- SEM) and of exogenous protein from 16 +/- 3 to 48 +/- 14 mmol. If fed continuously for 1 wk, a raw soybean diet caused endogenous protein loss to rise significantly from 221 +/- 26 to 432 +/- 85 mmol. We conclude that ingestion of food containing trypsin inhibitor affects nitrogen balance more by losses of amino acids of endogenous secreta than by losses of dietary amino acids.

Automated separation and measurement of urinary isoenzymes and protein by ion-exchange liquid chromatography.

This paper deals with a totally automated detection system for the assay of urinary isoenzymes and protein using high-performance liquid chromatography with a continuous post-column detection system. We attempted to determine the distribution of three enzymes in urine samples from a healthy child and in tissue extracts of rabbits. Alkaline phosphatase, gamma-glutamyltransferase and lactate dehydrogenase isoenzymes were each separated into six peaks. In comparison with the previous methods, this procedure provides better precision and accuracy, and it is sufficiently sensitive to allow the analysis without preconcentration of urine samples.

Identification of uncommon amino acids in the lentil seed (Lens culinaris Med.).

The fraction of the free basic amino acids in the lentil seed was shown to contain gamma-hydroxyarginine, gamma-hydroxyornithine and homoarginine besides the common amino acids. Similar distribution was found in 5 varieties of lentil, with hydroxyarginine and arginine dominating. The significance of these findings with regard to chemotaxonomy and lentil consumption is discussed.