NAT8 Variants, N-Acetylated Amino Acids, and Progression of CKD.

BACKGROUND AND OBJECTIVES: Genetic variants in NAT8, a liver- and kidney-specific acetyltransferase encoding gene, have been associated with eGFR and CKD in European populations. Higher circulating levels of two NAT8-associated metabolites, N-δ-acetylornithine and N-acetyl-1-methylhistidine, have been linked to lower eGFR and higher risk of incident CKD in the Black population. We aimed to expand upon prior studies to investigate associations between rs13538, a missense variant in NAT8, N-acetylated amino acids, and kidney failure in multiple, well-characterized cohorts. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We conducted analyses among participants with genetic and/or serum metabolomic data in the African American Study of Kidney Disease and Hypertension (AASK; n=962), the Atherosclerosis Risk in Communities (ARIC) study (n=1050), and BioMe, an electronic health record-linked biorepository (n=680). Separately, we evaluated associations between rs13538, urinary N-acetylated amino acids, and kidney failure in participants in the German CKD (GCKD) study (n=1624). RESULTS: Of 31 N-acetylated amino acids evaluated, the circulating and urinary levels of 14 were associated with rs13538 (P<0.05/31). Higher circulating levels of five of these N-acetylated amino acids, namely, N-δ-acetylornithine, N-acetyl-1-methylhistidine, N-acetyl-3-methylhistidine, N-acetylhistidine, and N2,N5-diacetylornithine, were associated with kidney failure, after adjustment for confounders and combining results in meta-analysis (combined hazard ratios per two-fold higher amino acid levels: 1.48, 1.44, 1.21, 1.65, and 1.41, respectively; 95% confidence intervals: 1.21 to 1.81, 1.22 to 1.70, 1.08 to 1.37, 1.29 to 2.10, and 1.17 to 1.71, respectively; all P values <0.05/14). None of the urinary levels of these N-acetylated amino acids were associated with kidney failure in the GCKD study. CONCLUSIONS: We demonstrate significant associations between an NAT8 gene variant and 14 N-acetylated amino acids, five of which had circulation levels that were associated with kidney failure.

Metabolic impact of pheochromocytoma/paraganglioma: targeted metabolomics in patients before and after tumor removal.

OBJECTIVE: Excess catecholamine release by pheochromocytomas and paragangliomas (PPGL) leads to characteristic clinical features and increased morbidity and mortality. The influence of PPGLs on metabolism is ill described but may impact diagnosis and management. The objective of this study was to systematically and quantitatively study PPGL-induced metabolic changes at a systems level. DESIGN: Targeted metabolomics by liquid chromatography-tandem mass spectrometry of plasma specimens in a clinically well-characterized prospective cohort study. METHODS: Analyses of metabolic profiles of plasma specimens from 56 prospectively enrolled and clinically well-characterized patients (23 males, 33 females) with catecholamine-producing PPGL before and after surgery, as well as measurement of 24-h urinary catecholamine using LC-MS/MS. RESULTS: From 127 analyzed metabolites, 15 were identified with significant changes before and after surgery: five amino acids/biogenic amines (creatinine, histidine, ornithine, sarcosine, tyrosine) and one glycerophospholipid (PCaeC34:2) with increased concentrations and six glycerophospholipids (PCaaC38:1, PCaaC42:0, PCaeC40:2, PCaeC42:5, PCaeC44:5, PCaeC44:6), two sphingomyelins (SMC24:1, SMC26:1) and hexose with decreased levels after surgery. Patients with a noradrenergic tumor phenotype had more pronounced alterations compared to those with an adrenergic tumor phenotype. Weak, but significant correlations for 8 of these 15 metabolites with total urine catecholamine levels were identified. CONCLUSIONS: This first large prospective metabolomics analysis of PPGL patients demonstrates broad metabolic consequences of catecholamine excess. Robust impact on lipid and amino acid metabolism may contribute to increased morbidity of PPGL patients.

Dietary intake of advanced glycation endproducts is associated with higher levels of advanced glycation endproducts in plasma and urine: The CODAM study.

BACKGROUND & AIMS: Advanced glycation endproducts (AGEs) are formed by the reaction between reducing sugars and proteins. AGEs in the body have been associated with several age-related diseases. High-heat treated and most processed foods are rich in AGEs. The aim of our study was to investigate whether dietary AGEs, are associated with plasma and urinary AGE levels. METHODS: In 450 participants of the Cohort on Diabetes and Atherosclerosis Maastricht study (CODAM study) we measured plasma and urine concentrations of the AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) using UPLC-MS/MS. We also estimated dietary intake of CML, CEL and MG-H1 with the use of a dietary AGE database and a food frequency questionnaire (FFQ). We used linear regression to investigate the association between standardized dietary AGE intake and standardized plasma or urinary AGE levels, after adjustment for age, sex, glucose metabolism status, waist circumference, kidney function, energy- and macro-nutrient intake, smoking status, physical activity, alcohol intake, LDL-cholesterol and markers of oxidative stress. RESULTS: We found that higher intake of dietary CML, CEL and MG-H1 was associated with significantly higher levels of free plasma and urinary CML, CEL and MG-H1 (ßCML = 0.253 (95% CI 0.086; 0.415), ßCEL = 0.194 (95% CI 0.040; 0.339), ßMG-H1 = 0.223 (95% CI 0.069; 0.373) for plasma and ßCML = 0.223 (95% CI 0.049; 0.393), ßCEL = 0.180 (95% CI 0.019; 0.332), ßMG-H1 = 0.196 (95% CI 0.037; 0.349) for urine, respectively). In addition, we observed non-significant associations of dietary AGEs with their corresponding protein bound plasma AGEs. CONCLUSION: We demonstrate that higher intake of dietary AGEs is associated with higher levels of AGEs in plasma and urine. Our findings may have important implications for those who ingest a diet rich in AGEs.

Enantioselective determination of citrulline and ornithine in the urine of d-amino acid oxidase deficient mice using a two-dimensional high-performance liquid chromatographic system.

Two-dimensional high-performance liquid chromatographic (2D-HPLC) and 2D-HPLC-mass spectrometric (2D-HPLC-MS) systems have been designed and developed for the determination of the citrulline (Cit) and ornithine (Orn) enantiomers. Several d-amino acids have already been identified as novel physiologically active molecules and biomarkers, and the enantioselective evaluation of the amounts, distributions and metabolisms of non-proteinogenic amino acids gain as well increasing interest. In the present study, highly selective analytical methods were developed using a capillary monolithic ODS column (0.53mm i.d. x 1000mm) for the reversed-phase separation of the target analytes from the matrix compounds in the first dimension, and a narrowbore-Pirkle type enantioselective column, KSAACSP-105S (1.5mm i.d. x 250mm), was used for the enantiomer separation in the second dimension. The amino acids were analyzed after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by the fluorescence detector and MS. The systems were applied to the urine of d-amino acid oxidase (DAO) deficient B6DAO- mice and control C57BL mice to evaluate the presence and metabolism of the Cit and Orn enantiomers in mammals. As a result, all of the 4 target enantiomers (d-Cit, l-Cit, d-Orn, l-Orn) were found in the urine of both strains. The %D value of Cit (d-Cit/Cit×100) increased about 3-fold in the urine of the DAO deficient mice and that of Orn also tended to increase with the DAO deficiency. These results were definitely confirmed by a 2D-HPLC-MS detection system. Further investigations about the biological significance of these d-isomers are currently ongoing.

High-throughput tandem mass spectrometry multiplex analysis for newborn urinary screening of creatine synthesis and transport disorders, Triple H syndrome and OTC deficiency.

BACKGROUND: Creatine synthesis and transport disorders, Triple H syndrome and ornithine transcarbamylase deficiency are treatable inborn errors of metabolism. Early screening of patients was found to be beneficial. Mass spectrometry analysis of specific urinary biomarkers might lead to early detection and treatment in the neonatal period. We developed a high-throughput mass spectrometry methodology applicable to newborn screening using dried urine on filter paper for these aforementioned diseases. METHODS: A high-throughput methodology was devised for the simultaneous analysis of creatine, guanidineacetic acid, orotic acid, uracil, creatinine and respective internal standards, using both positive and negative electrospray ionization modes, depending on the compound. RESULTS: The precision and accuracy varied by <15%. Stability during storage at different temperatures was confirmed for three weeks. The limits of detection and quantification for each biomarker varied from 0.3 to 6.3 µmol/l and from 1.0 to 20.9 µmol/l, respectively. Analyses of urine specimens from affected patients revealed abnormal results. Targeted biomarkers in urine were detected in the first weeks of life. CONCLUSIONS: This rapid, simple and robust liquid chromatography/tandem mass spectrometry methodology is an efficient tool applicable to urine screening for inherited disorders by biochemical laboratories.

Global host metabolic response to Plasmodium vivax infection: a 1H NMR based urinary metabonomic study.

BACKGROUND: Plasmodium vivax is responsible for the majority of malarial infection in the Indian subcontinent. This species of the parasite is generally believed to cause a relatively benign form of the disease. However, recent reports from different parts of the world indicate that vivax malaria can also have severe manifestation. Host response to the parasite invasion is thought to be an important factor in determining the severity of manifestation. In this paper, attempt was made to determine the host metabolic response associated with P. vivax infection by means of NMR spectroscopy-based metabonomic techniques in an attempt to better understand the disease pathology. METHODS: NMR spectroscopy of urine samples from P. vivax-infected patients, healthy individuals and non-malarial fever patients were carried out followed by multivariate statistical analysis. Two data analysis techniques were employed, namely, Principal Component Analysis [PCA] and Orthogonal Projection to Latent Structure Discriminant Analysis [OPLS-DA]. Several NMR signals from the urinary metabolites were further selected for univariate comparison among the classes. RESULTS: The urine metabolic profiles of P. vivax-infected patients were distinct from those of healthy individuals as well as of non-malarial fever patients. A highly predictive model was constructed from urine profile of malarial and non-malarial fever patients. Several metabolites were found to be varying significantly across these cohorts. Urinary ornithine seems to have the potential to be used as biomarkers of vivax malaria. An increasing trend in pipecolic acid was also observed. The results suggest impairment in the functioning of liver as well as impairment in urea cycle. CONCLUSIONS: The results open up a possibility of non-invasive analysis and diagnosis of P. vivax using urine metabolic profile. Distinct variations in certain metabolites were recorded, and amongst these, ornithine may have the potential of being used as biomarker of malaria. Pipecolic acid also showed increasing trend in the malaria patient compared to the other groups.

Gyrate atrophy of the choroid and retina with hyperornithinemia, cystinuria and lysinuria responsive to vitamin B6.

In this report, an 8-year-old girl is presented with the complaint of progressive night blindness. The authors have performed eye funduscopy, which showed chorioretinal atrophy in gyrate shape. A high level of plasma ornithine was determined. Urinary excretion of ornithine as well as lysine and cystine were increased. Patient was treated with high dose pyridoxine supplement (500 mg/dl). The night blindness condition of the patient improved. After 1 month of pyridoxine therapy ornithine level of her plasma was successfully reduced and blindness improved.

The L-arginine/NO pathway in end-stage liver disease and during orthotopic liver and kidney transplantation: biological and analytical ramifications.

The L-arginine/nitric oxide (L-Arg/NO) pathway is altered in liver and kidney diseases. However, the status of the L-Arg/NO pathway during and after orthotopic transplantation is insufficiently investigated and findings are uncertain because of analytical shortcomings. Also, most human studies have focused on individual members of the L-Arg/NO pathway such as nitrate or asymmetric dimethylarginine (ADMA). In the present article we report on a pilot study investigating extensively the status of the L-Arg/NO pathway before and during orthotopic liver transplantation (OLT). By using fully validated, highly sensitive and specific GC-MS and GC-MS/MS methods nitrite, nitrate, ADMA and its hydrolysis product dimethylamine (DMA), L-arginine and L-ornithine were measured in blood and urine. Our study gives strong evidence of the exceptional importance of hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity for the elimination of systemic ADMA. In end-stage liver disease the synthesis of NO and ADMA as well as the DDAH activity are elevated. However, increase in DDAH activity is insufficient to efficiently eliminate overproduced ADMA. The transplanted liver graft is capable of clearing ADMA in a rapid and sufficient manner. In contrast to studies from other groups, our study shows that in OLT as well as in living donor kidney transplantation, the second study reported here, reperfusion of the graft does not cause drastic alterations to the L-Arg/NO pathway with regard to NO synthesis. In the OLT study the concentration of circulating L-arginine fell temporally dramatically, while L-ornithine levels increased diametrically, most likely due to elevation of arginase activity. However, the relatively long-lasting decrease in plasmatic L-arginine in OLT seems not to have affected NO synthesis after reperfusion. Our OLT study suggests that liver reperfusion is associated with greatly elevated activity of proteolytic and hydrolytic enzymes including DDAH and arginase. Suppression of proteolytic and hydrolytic activity in transplantation could be a useful measure to improve outcome and remains to be investigated in further studies on larger patient collectives. The importance of analytical chemistry in this area of research is also discussed in this article.

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (HHH) presenting with acute fulminant hepatic failure.

We report on two Aboriginal patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome. Both presented with acute hepatic failure with severe hypertransaminasemia and coagulopathy, prompting evaluation for emergent liver transplantation. The diagnosis of HHH syndrome was based on the presence of typical metabolic abnormalities. A protein-restricted diet and L-arginine or L-citrulline supplementation were immediately started, with rapid normalization of liver function test results and other biochemical abnormalities. Molecular analysis of the SLC25A15 gene showed that the two patients were homozygous for the common French Canadian mutation (F188Delta). The diagnosis of HHH syndrome should be considered in patients with unexplained fulminant hepatic failure. There does not appear to be a genotype-phenotype correlation for this presentation, inasmuch as the only other reported patient presenting with this picture had two different point mutations. Early identification and prompt treatment of these patients is crucial to avoid liver transplantation and can be life saving.

HHH syndrome (hyperornithinaemia, hyperammonaemia, homocitrullinuria), with fulminant hepatitis-like presentation.

We report a 3-year-old Italian patient with the hyperornithinaemia, hyperammonaemia, homocitrullinuria (HHH) syndrome who presented with neurological deterioration after an intercurrent infection. Hyperammonaemia, coagulopathy and moderate hypertransaminasaemia were detected on hospital admission. Severe hepatocellular necrosis with hypertransaminasaemia (aspartate aminotransferase 20,000 UI/L, alanine aminotransferase 18,400 UI/L) and coagulopathy (PT < 5%) rapidly developed within few days, prompting evaluation for liver transplantation. A protein-restricted diet and arginine supplementation were immediately started, with a rapid improvement of the patient's neurological conditions and normalization of liver function tests and blood ammonia. The diagnosis of HHH syndrome was based on the presence of the typical metabolic abnormalities. Molecular analysis of the SLC25A15 gene showed that the patient was heterozygous for two novel mutations (G113C and M273K). The diagnosis of HHH syndrome should be considered in patients with fulminant hepatitis-like presentations. Early identification and treatment of these patients can be life-saving and can avoid liver transplantation.

Urinary excretion of total cystine and the dibasic amino acids arginine, lysine and ornithine in relation to genetic findings in patients with cystinuria treated with sulfhydryl compounds.

Advances in molecular genetics have brought a deeper understanding of cystinuria. This autosomal recessive disease, which is caused by a defective tubular reabsorption of cystine and the three dibasic amino acids arginine, lysine and ornithine, results in a lifelong risk of renal stone formation because of the low solubility of cystine in urine. Mutations detected within the two genes known to be associated with cystinuria, SLC3A1 (related to type I) and SLC7A9 (related to non-type I), cannot, however, in all cases explain the disease. Inasmuch as a high urinary concentration of cystine is the basis of stone formation in these patients, our aim was to measure urinary total cystine, arginine, lysine and ornithine, in patients currently lacking a full genetic explanation for their disease. Thirty-three patients with cystinuria who were on long-term treatment with tiopronin or D-penicillamine were divided into two groups. Group 1 comprised eight patients who carried mutation in one of the SLC3A1 alleles and two patients who completely lacked mutations both in the SLC3A1 and the SLC7A9 genes, that is genetic findings discordant with the increased urinary excretion of cystine and the dibasic amino acids in these patients. Group 2 comprised 23 patients homozygous for mutations within SLC3A1, that is genetic findings in accordance with the excretion pattern of classic type I cystinuria. When the two groups were compared, Group 1 had a significantly higher total urinary excretion of cystine ( p<0.01) as well as of arginine, lysine and ornithine ( p<0.05) than Group 2. Also, when the two patients without mutations were excluded from the calculations, there still was a significant difference in the urinary excretion of total cystine ( p<0.05). This suggests that the two patients without any detected mutations in the two known cystine transport genes also contributed to the difference. These unexpected findings indicate that an additional gene or genes participate in the urinary cystine reabsorption in the cystinuric patients who currently are without a full genetic explanation for their disease.

A mouse model for cystinuria type I.

Cystinuria, one of the most common inborn errors of metabolism in humans, accounts for 1-2% of all cases of renal lithiasis. It is caused by defects in the heterodimeric transporter system rBAT/b0,+AT, which lead to reduced reabsorption of cystine and dibasic amino acids through the epithelial cells of the renal tubules and the intestine. In an N-ethyl-N-nitrosourea mutagenesis screen for recessive mutations we identified a mutant mouse with elevated concentrations of lysine, arginine and ornithine in urine, displaying the clinical syndrome of urolithiasis and its complications. Positional cloning of the causative mutation identified a missense mutation in the solute carrier family 3 member 1 gene (Slc3a1) leading to an amino acid exchange D140G in the extracellular domain of the rBAT protein. The mouse model mimics the aetiology and clinical manifestations of human cystinuria type I, and is suitable for the study of its pathophysiology as well as the evaluation of therapeutic and metaphylactic approaches.

Cystinuria phenotyping by oral lysine and arginine loading.

BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acids transport that results in urolithiasis because of poor cystine solubility. Three cystinuria phenotypes, differentiated according to urinary amino acid excretion in obligate heterozygotes, were regarded as allelic variants of a monogenic disease. Two mutated amino acid transporter genes, however, have been recently identified as responsible for cystinuria. Mutations in the SLC3A1 gene. encoding for the heavy subunit of the transporter protein rBAT, were associated with type I cystinuria, whereas type II and III cystinuria were associated with mutations in the SLC7A9 gene, encoding for a light subunit of rBAT. Lysine and arginine metabolism have, therefore, been evaluated in cystinuria homozygotes and heterozygotes to better define the cystinuria phenotypes and their correlations with these emerging genotypes. PATIENTS AND METHODS: Lysine and arginine intestinal absorption and renal excretion were assessed by oral loading and compared to normal controls. Seven cystinuria homozygotes and 7 obligate heterozygotes belonging to the different types received alternately an oral dose of 0.5 mmol/kg body weight lysine or arginine. Plasma concentrations of lysine, arginine, ornithine (derived from rapid arginine conversion) were measured 0, 1, 2, and 3 hours after loading. Their urinary concentrations were measured in morning urine and in urine collected 0-6 hours after loading. RESULTS: Gut lysine absorption was deficient in type II and III, and normal in type I cystinuria homozygotes. Impaired arginine intestinal absorption, as well as massive lysine, arginine, and ornithine hyperexcretion were shared by all homozygotes, irrespective of the type. All heterozygotes shared normal lysine absorption, whereas arginine absorption was slightly impaired in type II and III heterozygotes, which also displayed high lysine, arginine, and ornithine urinary excretion after loading. CONCLUSIONS: Two cystinuria phenotypes, type I and non-type I, can be identified in both homozygous and heterozygous cystinuric subjects by oral loading tests with lysine and arginine. In agreement with recent molecular findings, non-type I cystinuria comprises mentioned type II and type III, which constitute allelic variants of a cystine and dibasic amino acid transport disorder distinct from type I cystinuria.

Liquid chromatographic determination of ornithine and lysine based on intramolecular excimer-forming fluorescence derivatization.

A highly sensitive and selective fluorometric determination method for ornithine and lysine has been developed. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase liquid chromatography (LC). The analytes, containing two amino moieties in a molecule, were converted to the corresponding dipyrene-labeled derivatives by reaction with PSE. The derivatives afforded intramolecular excimer fluorescence (450-550 nm) which can clearly be discriminated from the normal fluorescence (370-420 nm) emitted from PSE and monopyrene-labeled derivatives of monoamines. The structures of the derivatives and the emission of excimer fluorescence were confirmed by LC with mass spectrometry and with three-dimensional fluorescence detection system, respectively. The PSE derivatives of ornithine and lysine could be separated by reversed-phase LC on ODS column with isocratic elution. The detection limits (signal-to-noise ratio = 3) for ornithine and lysine were 3.5 and 3.7 fmol, respectively, for a 20-microl injection. Furthermore, this method had enough selectivity and sensitivity for the determination of ornithine and lysine in normal human urine.

Possible role of polyamines in gyrate atrophy.

PURPOSE: Gyrate atrophy (GA) is marked by hyperornithinemia and lowered ornithine amino transferase (OAT). However there are patients of GA without hyperornithinemia and those with hyperornithinemia without GA. Some cases of GA have been reported to have low lysine. The purpose of the study was to determine if polyamines, the metabolites of ornithine, and lysine have any diagnostic role in GA. METHODS: Ornithine in plasma was estimated by two-dimensional paper chromatography, with elution of the coloured spot, and the absorbance measured using a spectrophotometer at 560 nm. OAT assay in lymphocytes was done spectrophotometrically using ornithine as substrate. Blood and urinary polyamines were extracted with n-butanol, benzoylated and analysed with HPLC; putrescine, spermine, spermidine, and cadaverine were assayed individually at 254 nm with the UV detector using ODS, G18 column with 63% methanol as solvent. RESULTS: Of the 7 patients investigated, 6 had features typical of GA. One was diagnosed to have atypical retinitis pigmentosa (case 3). The first five cases had elevated ornithine and diminished OAT, but cases 6 and 7 had near-normal ornithine and case 7 had near-normal OAT. However, all 7 patients had increased levels of total polyamines in urine compared to normals. Five had increased putrescine and three had increased spermine. All the 7 had decreased cadaverine in urine. Thus, though there were inconsistencies with ornithine and OAT, all the 7 patients had elevated polyamines from ornithine and decreased cadaverine. CONCLUSION: In addition to estimating ornithine and OAT in GA, it is suggested that urinary polyamines may be analysed as the latter appears to correlate better with the clinical condition and help in the diagnosis to a greater extent. Moreover, while ornithine is an innocuous amino acid, polyamines are known to damage DNA and proteins.

[Phenotypical manifestations of cystinuria: a study of 20 families in the Community of Valencia]. / Manifestaciones fenotípicas de la cistinuria: estudio de 20 familias en la Comunidad Valenciana.

BACKGROUND: The aim of this study was to classify phenotypically cystinuria patients in the Comunidad Valenciana using their genealogy and to study the large heterogeneity of the disease expression. SUBJECTS AND METHOD: From 29 patients diagnosed of cystinuria, 20 families were enrolled in the study. An urine sample of every subject was collected to quantify urine amino acids using high pressure liquid chromatography. We also looked for the presence of cystine crystals. Genetic analyses were carried out using PCR (Polymerase Chain Reaction), and RFLPs (Restriction Fragment Length Polymorphisms). Demographic and clinical characteristics were recorded in a standard questionnaire. RESULTS: From 20 families, 4 were classified Type I, 10 as Type non I, and 6 as Type unknown. Type I cystinuria patients showed the highest urinary levels of cystine and ornithine. We also found an association of cystine crystals and M467T mutation in SLC3A1 gene with Type I. However, we did not find a higher risk of nephrolithiasis associated to any family type. CONCLUSIONS: Phenotypical characterization of patients with cystinuria has showed the wide variability of phenotypical traits indeed in the same transmission pattern. This variability could be due to the genetic and environmental heterogeneity which has developed this pathology and modulated its evolution.

Plasma concentrations and renal clearance of orotic acid in argininosuccinic acid synthetase deficiency.

Argininosuccinic acid synthetase deficiency (ASD) is a rare disorder of urea cycle metabolism, with pronounced citrullinemia and orotic aciduria being characteristic biochemical features. To further investigate the role of plasma orotic acid and its possible use for monitoring the metabolic status in ASD, we determined plasma orotic acid, amino acid, and ammonium levels in plasma samples collected over a period of 3 years from a patient who is now 8 years of age. Orotic acid plasma concentrations varied widely from less than 1 micromol/l to more than 60 micromol/l. The renal clearance of orotic acid was eightfold the glomerular filtration rate, thus supporting an active mechanism underlying the excretion of this pyrimidine. Data obtained during a metabolic crisis yielded a statistically significant linear correlation of orotic acid plasma levels with those of glutamine and ammonium, which are generally accepted for assessment of the successful treatment of this disorder. Our data revealed no advantage of plasma orotic acid concentrations over the established amino acids (glutamine and arginine) and ammonium for determining acute treatment responses. Since several effects of high levels of orotic acid have been described in mammals, further research is necessary to assess a possible contribution of orotic acid to the pathogenesis of ASD and the use of plasma orotic acid levels in the long-term monitoring of these patients.

Reduced nitric oxide production by L-arginine deficiency in lysinuric protein intolerance exacerbates intravascular coagulation.

Lysinuric protein intolerance (LPI) results in low serum L-arginine, hyperammonemia, mental retardation, thrombocytopenia, and an increased frequency of bowel movements. Our objective was to evaluate the effects of low serum L-arginine, the essential substrate for reactions catalyzed by nitric oxide synthetase (NOS), on the serum nitric oxide (NO) level and coagulation activity in a patient with LPI. A 37-year-old Japanese man who presented with abdominal pain and subnormal fasting levels of serum L-arginine and L-lysine was found to have LPI. The result of oral administration of diamino acids was an increased in urine and a decrease in serum, thus confirming the diagnosis. A decrease in the platelet count and an increase in the plasma levels of thrombin-antithrombin III complex (TAT) and fibrin degradation products (FDPs) indicated the presence of subclinical intravascular coagulation. Serum levels of NO derivatives and L-arginine were determined after intravenous administration of L-arginine. The effects of intravenous L-arginine or transdermal nitroglycerin on the plasma level of TAT were also investigated. Serum levels of NO derivatives were significantly reduced in the LPI patient versus the healthy control group (n = 5). Intravenous administration of L-arginine increased the serum level of NO derivatives and the platelet count and reduced plasma TAT and FDP levels. The plasma level of TAT was also reduced by transdermal nitroglycerin. A decrease in the serum level of L-arginine in patients with LPI appears to result in a decrease in NO production. The improvement in plasma TAT levels produced by administration of intravenous L-arginine or transdermal nitroglycerin suggests that intravascular coagulation is exacerbated by the decrease of NO production in patients with LPI.

Reference values of urinary excretion of cystine and dibasic aminoacids: classification of patients with cystinuria in the Valencian Community, Spain.

OBJECTIVE: Cystinuria is an autosomal-recessive disorder of the kidneys and small intestine affecting a luminal transport mechanism shared by cystine, ornithine, arginine, and lysine. Three different types of cystinuria can be distinguished according to the excretion of these amino acids in urine samples. We propose cutoff values from our population as references and we present a classification of cystinuric patients using quantitative amino acid chromatography in first morning urine samples. DESIGN AND METHODS: A random sample of forty healthy subjects belonging to general population of the Valencian Community were selected as control subjects. Cystine, lysine, arginine, and ornithine were quantified by reverse-phase HPLC. Seventy-two subjects, diagnosed previously as cystinuric by the cyanide-nitroprusside test were classified. Probands excreting more than 113.12 micromol cystine per mmol of creatinine (i.e., 1,000 micromol cystine per gram of creatinine) were classified as homozygotes. Parents of homozygotes in whom excretion of amino acids were normal were classified as heterozygotes type I. Those probands showing the excretion of at least one amino acid and the sum of urinary cystine plus the basic amino acids higher than the corresponding references ranges in our population were classified as heterozygotes type II or type III (heterozygotes non-type 1). RESULTS: We identified 24 homozygotes, 39 non-type I heterozygotes and 3 type I heterozygotes. The remaining 6 probands could not be classified. Means for cystine, lysine, arginine ornithine and their sum in homozygotes and heterozygotes non-type I were significantly (p < 0.001) in excess of the respective reference ranges. Moreover, means values in homozygotes were statistically different (p < 0.001) from heterozygotes non-type I. CONCLUSION: Urinary excretion of cystine per mmol creatinine allow us to distinguish heterozygotes from homozygotes. However, the best discriminator to distinguish non-type I heterozygotes from normal population might be the excretion of lysine per mmol creatinine. Additional studies including characterization of appropriate haplotypes should be carried out for a more precise identification of types of cystinuria.