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.

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.

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.

[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.

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.

Prospective analysis and classification of patients with cystinuria identified in a newborn screening program.

Patients who inherit mutant cystinuria genes excrete high concentrations of cystine, ornithine, arginine, and lysine in the urine. At least three variants of cystinuria can be distinguished in heterozygotes. To determine whether certain combinations of mutant genes are more disadvantageous than others, we analyzed amino acid excretion in families of 17 probands with cystinuria identified by the Quebec neonatal screening program. Parents of the probands were classified into the three known phenotypes by calculating the sum of cystine, ornithine, arginine, and lysine excretion. Although parents of type I/I homozygotes excreted amounts of cystine in the normal range, their offspring excreted significantly greater amounts of urinary cystine than did children who have type I/III genetic compounds. This observation suggests that types I and III cystinuria mutations might involve two distinct genetic loci. Children with type I/I homozygous cystinuria often excrete cystine at levels greater than the theoretic solubility limit and may be at greatest risk for nephrolithiasis. We outline an approach to monitoring children with cystinuria who come to medical attention before formation of cystine stones.

Bardet-Biedl syndrome and cystinuria.

An unusual association of Bardet-Biedl syndrome with cystinuria was described in one patient. A 21-year-old male was admitted to hospital because of renal failure, severe deterioration of visual acuity, polydactyly, brachydactyly, and mental retardation. Laboratory investigations revealed a serum creatinine of 292 mumol/L (3.3 mg/dL) and a GFR of 25 mL/min per 1.73 m2. Quantitative ion exchange chromatography demonstrated an increased urinary excretion rate of cystine, lysine, arginine, and ornithine. The ophthalmologic examination showed a severe atypical retinal dystrophy. Visual acuity was severely deteriorated and the patient could only count the examining physician's fingers. The patient had been previously evaluated at the age of 7 years for polyuria, polydipsia, and growth failure. His workup at that time demonstrated nephrogenic diabetes insipidus, normal GFR, and a urinary amino acid pattern consistent with the cystinuric phenotype. There was mental retardation notwithstanding the normal ophthalmologic examination. Intravenous pyelography showed calyceal clubbing, calyceal cysts, and lobulated renal outlines of the fetal type. The patient was evaluated again at the age of 13 years for deterioration of visual acuity and the ophthalmologic examination showed an atypical retinal dystrophy, with sparse pigmentation, central and peripheral atrophy, attenuated vessels, and marked optic disk pallor. To our knowledge the association of Bardet-Biedl syndrome with cystinuria has never been reported. It is unlikely that cystinuria may have contributed to the kidney damage. The possibility that mental retardation has been induced or aggravated by cystinuria cannot be excluded.

Cerebellar ataxia with glutamic aciduria.

We report a case of cystinuria and glutamic aciduria, presenting with progressive cerebellar manifestations. She had cerebellar type dysarthria and limb ataxia. Head MRI revealed cerebellar atrophy. Urinary amino acid analysis showed excessive excretion of glutamate and the dibasic amino acids (cystine, arginine, lysine, and ornithine). Cystine and glutamic acid are thought to be transported in a common membrane transport system. Reduction of glutamic acid and cystine in the cerebrospinal fluid was revealed. A relationship between cystinuria and cerebellar manifestation was discussed.

Diagnostic and genetic studies in 43 patients with classic cystinuria.

We present results for laboratory screening and diagnostic tests--cyanide-nitroprusside test, semi-quantitative thin-layer chromatography, and quantitative amino acid column chromatography--of 43 patients with classic cystinuria. We report the efficaciousness of the cyanide-nitroprusside test and of thin-layer chromatography, as compared with quantitative amino acid chromatography, for detecting heterozygotes for type II or III cystinuria. The quantitative results for aminoaciduria in 57 blood relatives in 23 families were used to categorize the index patients with classic cystinuria. By column chromatography the ranges of excretion rates (mumol/24 h per 1.73 m2 body surface area) of diagnostic amino acids in the index patients were as follows: cystine 556-54044 (normal 20-128), arginine 131-11543 (10-80), lysine 768-21848 (51-514), ornithine 185-5685 (0-80). Also by column chromatography the median values for arginine and ornithine excretion in cystine-lysinuric heterozygotes (among the 57 blood relatives) were significantly higher (P less than 0.01) than in controls but never approached the values for homozygotes. The cyanide-nitroprusside test results were positive in urine samples of 41 of 43 index patients and in 16 (51.6%) of the urine samples of 31 obligate heterozygotes with column chromatographically proven cystine-lysinuria. Thin-layer chromatography detected all of the homozygotes, all the compound heterozygotes, and 54.8% of the carriers. According to the type of aminoaciduria in their relatives, 11 patients with classic cystinuria could be classified as having classic cystinuria type I, 11 as having type II or III, and three as being compound heterozygotes. We discuss the implications of these results for correct diagnoses and for genetic studies in classic cystinuria.

Effects of variation in sodium intake on cystinuria.

This study was designed to characterize the effect of urinary sodium excretion on amino acid excretion in cystinuria. We studied three homozygous patients with cystinuria at various levels of sodium intake ranging between 50 and 230 mmol/day. A reduction in sodium intake produced a marked decrease in rates of urinary cysteine excretion. The low-sodium diet also reduced the excretion of lysine but had no significant effect on the excretion of ornithine or arginine. We conclude that a dietary restriction of sodium may reduce the urinary excretion of cystine and can provide a simple and safe long-term approach to the treatment of cystinuria.

Urinary arginine and ornithine in occupational exposure to 2-ethylhexanoic acid.

Nine sawmill workers were divided into two groups according to their exposure to 2-ethylhexanoic acid, (EHA), a pesticide which has replaced the older pentochlorophenol. The men with lower exposure excreted 30 +/- 10 nmol EHA/mmol creatinine (mean +/- SD, n = 4) in urine samples taken after the workshift, whereas men with higher exposure excreted 1.8 +/- 1.6 mumol EHA/mmol creatinine (mean +/- SD, n = 5, p less than 0.01). The urinary ornithine and arginine concentrations were at the lower exposure 1.4 +/- 0.4 and 1.5 +/- 0.8 mumol/mmol creatinine, respectively (mean +/- SD, n = 4), and they increased significantly (p less than 0.01) to 4.5 +/- 2.5 and 3.2 +/- 1.5 mumol/mmol (mean +/- SD, n = 5), respectively, at the higher exposure. This might have been caused by the inhibitory effect of EHA on urea synthesis which was partially compensated for by elevated arginine and ornithine concentrations to drive the urea cycle more efficiently.

The effect of L-glutamine and sodium intake in cystinuric patients.

Four patients with cystinuria were studied to assess the effect of L-glutamine and sodium on the urinary excretion of dibasic amino acids. L-Glutamine 2.1 g/d and sodium about 300 mmol/d were given for three weeks, after which L-glutamine 2.1 g/d and sodium about 150 mmol/d were given for a further three weeks. L-Glutamine reduced the excretion of dibasic amino acids in one patient with both sodium regimens, but not significantly. In the remaining three patients no reduction was observed.

Epiretinal membranes and cystoid macular edema in gyrate atrophy of the choroid and retina.

A 26-year-old woman with gyrate atrophy of the choroid and retina and hyperornithinemia lost central visual acuity because of cystoid macular edema associated with epiretinal membrane formation. Dietary manipulation over a 5-month interval reduced the plasma ornithine levels 37% but did not halt the progression of the gyrate atrophy nor influence the cystoid edema or visual acuity.

Anticystinuric effects of glutamine and of dietary sodium restriction.

We studied four patients with cystinuria to assess the effects of glutamine and dietary sodium on the urinary excretion of dibasic amino acids. In Patient 1, at an ad libitum dietary sodium intake of about 300 mmol per day, oral administration of glutamine led to reproducible and marked anticystinuria and antiornithinuria, whereas the excretion of lysine and arginine was not significantly affected. In Patient 2, at an ad libitum dietary sodium intake of about 150 mmol per day, no effect of glutamine could be demonstrated in studies lasting up to three weeks. Since the principal difference between Patients 1 and 2 was their dietary intake of sodium, Patient 3 was studied during dietary sodium intakes of 150 and 300 mmol per day. His cystine excretion was found to be higher at 300 than at 150 mmol per day. Glutamine suppressed his cystine excretion at a sodium intake of 300 mmol per day but had no effect at 150 mmol per day. When the effect of a further reduction in sodium intake alone was studied in a fourth patient, a decrease of 150 to 50 mmol per day was found to reduce cystine excretion markedly within 17 days. The low-sodium diet alone also reduced the excretion of lysine, arginine, and ornithine. We conclude that glutamine may reduce the excretion of dibasic amino acids at a high sodium intake but not at an intake of about 150 mmol per day. However, since a sodium-dependent excretion of the dibasic amino acids occurs at an intake down to about 50 mmol of sodium per day, dietary restriction of sodium can provide a safe approach to the treatment of cystinuria.

Heterozygous cystinuria and urinary lithiasis.

Cystinuria is a recessively inherited transport disorder, with at least three mutant alleles (I, II, and III) demonstrable. I/I, II/II, and III/III homozygotes and I/II, I/III, and II/III compound heterozygotes (cystinuric patients) have high urinary concentrations of cystine, lysine, arginine, and ornithine and frequently form cystine stones. +/I heterozygotes (nondetectable) are phenotypically normal, whereas +/II and +/III heterozygotes (detectable) show variable increases in urinary cystine and lysine concentration and at times increases in urinary arginine levels. The objectives of the present study were to determine the frequency of +/II heterozygotes among stone-forming and nonstone-forming individuals from the same region of Brazil and to evaluate the possible relationship between heterozygous cystinuria and urinary lithiasis. When urine samples from 5,150 individuals (5,000 nonstone-forming individuals and 150 stone-forming individuals) were screened by the qualitative cyanide-nitroprusside cystine test, by thin-layer amino acid chromatography, and by quantitative amino acid determination by ion-exchange chromatography, 32 +/II or +/III heterozygotes (26 nonstone-forming and six stone-forming individuals) were detected. The frequency of detectable heterozygotes among the stone-forming individuals (1:25) was significantly higher than that among nonstone-forming individuals (1:104), which provides additional evidence that heterozygosity for +/II and +/III cystinuria is a risk factor in the formation of urinary stones. No significant difference was detected in urinary cystine concentration or in terms of the various characteristics of urolithiasis when stone-forming heterozygotes were compared to nonstone-forming heterozygotes. These data suggest that the tendency towards stone-forming among heterozygotes is probably owing to a complex and multifactorial mechanism.

Ontogeny modifies manifestations of cystinuria genes: implications for counseling.

Among 339,868 newborn infants screened at 3 weeks of age (91% compliance rate), 730 had elevated rates of excretion of cystine and the dibasic amino acids lysine, ornithine, and arginine; 191 infants had persistent "infantile cystinuria" on follow-up screening (100% compliance). Apparent incidence of the phenotype was 562 per million infants; this rate is seven times higher than for classic cystinuria in the adult segment of the Quebec population. We studied longitudinally 26 probands 2 to 4 months of age. Initially, each excreted cystine and dibasic amino acids at much higher levels than did normal infants or either parent. From parental phenotypes (heterozygous or homozygous normal) and urine amino acid excretion values at 6 months of age in probands, the infants were classified as either heterozygous for the various classic cystinuria genotypes--type I ("silent"), eight infants; type II (high excretor), three; type III (moderate excretor), nine--or homozygous (and genetic compound), six. Urine amino acid excretion diminished steadily with age, to reach the variant parental value in heterozygous infants but not in homozygotes. Cystinuria heterozygotes, with the possible exception of some type I individuals, could not be distinguished reliably from homozygotes in early infancy, although homozygotes had significantly higher excretion values as a group. We deduce that renal ontogeny amplifies phenotypic expression of cystinuria alleles, thus influencing correct classification of genotype (heterozygote vs homozygote, and type of allele). These findings have implications for counseling and the need for follow-up of infantile cystinuria.