Analysis Of Amino Acids In Urine Research Articles

Dried urine spot (DUS) applied for sampling prior to the accurate HILIC-MS/MS determination of 14 amino acids

Urine amino acid analysis has proven valuable for an array of clinical or nutritional studies. However, transportation of liquid urine sample shows certain disadvantages, such as possible leakage, need for cold chain and thus higher costs for their transport. Utilization of dried urine spots (DUS) can offer an interesting alternative. In the present study, a method was developed for the determination of 14 amino acids in DUS including the testing of in-house collection device and drying of the sample before analysis. Normal filter paper was tested as the means for sample collection. Absorption and extraction experiments were performed on 3 different types of filter paper, with 3 different extraction solvents and two different solvent volumes. The solvents used were mixtures of common analytical solvents (methanol, water, acetonitrile) using total volumes of 1 mL and 1.5 mL. Finally, 1 mL of acetonitrile: methanol: water 40:40:20 (v/v/v) was chosen as the optimal system. Analysis was performed on a UHPLC-MS system, using stable isotope labeled internal standards. Method validation included the study of limits of detection (LOD) and quantification (LOQ), linearity ranges, precision, matrix effect, extraction recovery, precision, and stability for each analyte. The obtained results were satisfactory, thus enabling application of the proposed method as an alternative to the analysis of liquid urine. Further utilization of DUS can offer advantages by enabling patient centric sampling even in long distances far from the analytical laboratories.

A-326 Argininosuccinic Acid - is it Routinely Detectable in the Urine of Healthy Individuals?

Abstract Background Argininosuccinic aciduria is a genetic disorder that results in elevated levels of argininosuccinic acid (ASA) in urine and blood. While there is seemingly a consensus within the clinical literature that any detectable amount of ASA in blood/plasma is diagnostic for argininosuccinic aciduria, yet there is conflict as to whether ASA in urine can be found in unaffected individuals. Some literature states that ASA should not be found in the urine of a healthy individual, while other literature reports low levels of ASA in the urine of healthy individuals. Additionally, many laboratories do not have a normal range for ASA within urine, which implies that any level of ASA found within urine is indicative of argininosuccinic aciduria. This conflict may in part be due to differences in the methods used to determine ASA levels. Newer liquid chromatography tandem mass spectrometry (LCMSMS) methods have been shown to be more sensitive than ion-exchange chromatography (IEC) based amino acid methods for detection of ASA. Additionally, the detection of ASA via IEC requires the heating and acidification of the sample to convert ASA into a single peak for quantitation. Heating and acidification is not routinely done for urine amino acid analysis with IEC; with routine IEC urine amino acid analysis, ASA migrates as three small peaks that may go unnoticed. In our clinical laboratory, which utilizes the less sensitive IEC method, our procedure describes the presence of any ASA in urine as diagnostic for argininosuccinic aciduria. Given the conflicting literature, we set forth to determine whether low levels can be detected in unaffected individuals with an IEC method, and if so, establish a normal range of ASA levels in urine with IEC. Methods Twenty waste urine samples from patients with no prior diagnoses of argininosuccinic aciduria were evaluated. ASA levels within the samples were determined via high performance ion exchange chromatography for separation of compounds followed by derivatization with ninhydrin for spectrophotometric detection with a Hitachi Amino Acid analyzer. The samples were acidified and heated, which converts ASA into a single anhydride peak for accurate quantitation, and the results were normalized to urine creatinine. Results ASA was detected in all twenty urine samples, with an average ASA concentration of 27.2 μmol/g Cr and a range of 11.3–47.7 μmol/g Cr. Conclusion Given these findings, our clinical laboratory procedure has been updated to include the information that ASA can be present in the urine of healthy individuals up to a concentration of 50 μmol/g Cr. Other clinical laboratories that analyze urine to identify argininosuccinic aciduria should be aware of the misinformation in the literature that claims that ASA is not present in the urine of healthy individuals.

Cystine and amino acid concentrations in the urine of pet ferrets (Mustela putorius furo)

The prevalence of cystine urolithiasis in domestic ferrets (Mustela putorius furo) has increased dramatically in the past decade. Genetic mutations associated with a defect in the active resorption of cysteine (which is oxidized to cystine) and dibasic amino acids in the proximal renal tubules leading to cystinuria were described in several species; however, the etiology in ferrets is unknown. This study aimed to characterize cystinuria in pet ferrets. Urine samples were aseptically acquired from 27 domestic client-owned ferrets with known dietary and medical histories. The samples underwent a routine urinalysis and microscopic evaluation. Urine aliquots were deproteinized with 6% sulfosalicylic acid and analyzed for amino acid concentrations by ion exchange chromatography. Urine cystine concentrations ranged from 8 to 2486 nmol/mL (median 243; SD 540.02). Principal component analyses revealed correlations among urinary amino acid concentrations, which were explained mostly by three components. Analyses revealed significant correlations between cystine and citrulline, ornithine, and lysine concentrations. Linear regression revealed a negative correlation between age and urinary cystine concentrations and no significant correlation between cystinuria and diet or treats. This is the 1st description of urine amino acid analysis in a cohort of pet ferrets. The detected correlations between cystine, ornithine, and citrulline could indicate that the pathology responsible for cystine urolithiasis in ferrets is like in other species. Result may indicate young ferrets are at increased risk of cystine urolithiasis Further research is needed to evaluate the possible association between diet and cystinuria.

Chapter 11 - Laboratory and metabolic investigations

Clinical variability and substantial overlap between mitochondrial disorders and other genetic disorders and inborn errors make the clinical and metabolic diagnosis of mitochondrial disorders quite challenging. Evaluating specific laboratory markers is essential in the diagnostic process, but mitochondrial disease can be present in the absence of any abnormal metabolic markers. In this chapter, we share the current consensus guidelines for metabolic investigations, including investigations in blood, urine, and the cerebral spinal fluid and discuss different diagnostic approaches. As personal experience might significantly vary and there are different recommendations published as diagnostic guidelines, the Mitochondrial Medicine Society developed a consensus approach based on literature review for metabolic diagnostics in a suspected mitochondrial disease. According to the guidelines, the work-up should include the assessment of complete blood count, creatine phosphokinase, transaminases, albumin, postprandial lactate and pyruvate (lactate/pyruvate ratio when the lactate level is elevated), uric acid, thymidine, amino acids, acylcarnitines in blood, and urinary organic acids (especially screening for 3-methylglutaconic acid). Urine amino acid analysis is recommended in mitochondrial tubulopathies. CSF metabolite analysis (lactate, pyruvate, amino acids, and 5-methyltetrahydrofolate) should be included in the presence of central nervous system disease. We also suggest a diagnostic strategy based on the mitochondrial disease criteria (MDC) scoring system in mitochondrial disease diagnostics; evaluating muscle-, neurologic-, and multisystem involvement, and the presence of metabolic markers and abnormal imaging. The consensus guideline encourages a primary genetic approach in diagnostics and only suggests a more invasive diagnostic approach with tissue biopsies (histology, OXPHOS measurements, etc.) after nonconclusive genetic testing.

Clinical Reasoning: A Young Adult Man With Cognitive Changes, Gait Difficulty, and Renal Insufficiency.

A 22-year-old right-handed man with recently diagnosed gout and renal insufficiency presented with 3 months of progressive gait instability and cognitive changes. He initially presented to an outside institution and underwent a broad workup, but an etiology for his symptoms was not found. On subsequent presentation to our institution, his examination revealed multidomain cognitive dysfunction, spasticity, hyperreflexia, and clonus. A broad workup was again pursued and was notable for an MRI of the brain, revealing cortical atrophy advanced for his age, bland CSF, and a weakly positive serum acetylcholine receptor ganglionic neuronal antibody of unclear significance. The history of gout and inadequately explained renal insufficiency led to a workup for inborn errors of metabolism, including urine amino acid analysis, which revealed a homocysteine peak. This finding prompted further evaluation, revealing markedly elevated serum homocysteine and methylmalonic acid and low methionine. He ultimately developed superficial venous thromboses, a segmental pulmonary embolism, and clinical and electrographic seizures. He was initiated on appropriate treatment, and his symptoms markedly improved. The case serves as a reminder to include late-onset inborn errors of metabolism in the differential for young adult patients with onset of neurologic, psychiatric, renal, and thromboembolic symptoms.

Acquired transient Fanconi syndrome after feeding jerky treats in dogs - A case series

Between April 2018 and August 2019, 6 dogs with laboratory diagnostic evidence of Fanconi syndrome were presented to the Tierklinik Hofheim. The presumptive diagnosis was confirmed via urine amino acid analysis in all dogs. In 5 of the 6 dogs an alimentary origin was suspected, in 3 dogs the course of the disease could be followed. Supportive therapeutic measures and elimination of jerky treats from the dogs' diet improved the clinical symptoms in all dogs with known follow-up. This is the first case series of dogs with alimentary acquired Fanconi syndrome in Germany. The frequency of occurrence of the disease in that short period of time in only one small animal clinic suggests a significantly higher incidence than currently assumed.

Lysinuric protein intolerance: Pearls to detect this otherwise easily missed diagnosis.

BACKGROUND:Lysinuric protein intolerance (LPI) is a rare autosomal recessive disorder characterized by deficient membrane transport of cationic amino acids. It is caused by pathogenic variants in SLC7A7, resulting in impairment of intestinal import and renal proximal tubule loss of the affected amino acids. LPI typically presents with gastrointestinal symptoms, such as vomiting, diarrhea, and failure to thrive.CASE REPORT:A 4-year-old African-American boy presented with multiple respiratory tract infections, weight loss in the setting of chronic diarrhea and worsening abdominal distention, and multiple episodes of rectal prolapse. Development was unaffected. Laboratory examination demonstrated mild anemia, hypokalemia and hypoalbuminemia, transaminitis, and normal ammonia. Initial urine amino acid analysis did not show major elevations of lysine and ornithine, often lower than expected in the setting of malnutrition. Upon initiation of total parenteral nutrition (TPN), his urine amino acids showed a characteristic profile of dibasic aminoaciduria.CONCLUSIONS:Failure to thrive, chronic diarrhea, and hepatomegaly should raise suspicion for LPI. Urine amino acids can be normal in this condition in the setting of malnutrition, a common complication of the disease. Additionally, it has been previously shown that the plasma arginine and ornithine concentration is higher in LPI subjects.

Clinical, biochemical, mitochondrial, and metabolomic aspects of methylmalonate semialdehyde dehydrogenase deficiency: Report of a fifth case

Methylmalonate semialdehyde dehydrogenase deficiency (MMSDD; MIM 614105) is a rare autosomal recessive defect of valine and pyrimidine catabolism. Four prior MMSDD cases are published. We present a fifth case, along with functional and metabolomic analysis. The patient, born to non-consanguineous parents of East African origin, was admitted at two weeks of age for failure to thrive. She was nondysmorphic, had a normal brain MRI, and showed mild hypotonia. Gastroesophageal reflux occurred with feeding. Urine organic acid assessment identified excess 3-hydroxyisobutyrate and 3-hydroxypropionate, while urine amino acid analysis identified elevated concentrations of β-aminoisobutyrate and β-alanine. Plasma amino acids showed an elevated concentration of β-aminoisobutyrate with undetectable β-alanine. ALDH6A1 gene sequencing identified a homozygous variant of uncertain significance, c.1261C > T (p.Pro421Ser). Management with valine restriction led to reduced concentration of abnormal analytes in blood and urine, improved growth, and reduced gastroesophageal reflux. Western blotting of patient fibroblast extracts demonstrated a large reduction of methylmalonate semialdehyde dehydrogenase (MMSD) protein. Patient cells displayed compromised mitochondrial function with increased superoxide production, reduced oxygen consumption, and reduced ATP production. Metabolomic profiles from patient fibroblasts demonstrated over-representation of fatty acids and fatty acylcarnitines, presumably due to methylmalonate semialdehyde shunting to β-alanine and subsequently to malonyl-CoA with ensuing increase of fatty acid synthesis. Previously reported cases of MMSDD have shown variable clinical presentation. Our case continues the trend as clinical phenotypes diverge from prior cases. Recognition of mitochondrial dysfunction and novel metabolites in this patient provide the opportunity to assess future patients for secondary changes that may influence clinical outcome.

Acquired Fanconi syndrome in a dog exposed to jerky treats in Japan

A 6-year-old spayed female Jack Russell Terrier presented with a 1-month history of lethargy, anorexia, vomiting and weight loss. The dog was fed beef and chicken jerky treats daily in addition to a commercial diet. Laboratory tests revealed azotemia, hypokalemia, hyperchloremia, metabolic acidosis and glucosuria with normoglycemia. Urine amino acid analysis showed significant amino acid loss into the urine. Thus, Fanconi syndrome was diagnosed, and based on the case history and extensive diagnostic testing, excessive consumption of jerky treats was strongly suspected as the cause. Glucosuria resolved 7 days after the withdrawal of jerky treats and fluid therapy. Aminoaciduria was substantially, but not completely, improved 3 months after diagnosis. Mild azotemia remained, suggesting chronic renal disease. To the best of our knowledge, this is the first reported case of Fanconi syndrome following the consumption of jerky treats in Japan.

Two Japanese brothers with hereditary γ‐glutamyl transpeptidase deficiency

We report on two Japanese brothers with hereditary deficiency in gamma-glutamyl transpeptidase. The propositus was 48 years old when he first visited our medical center and had a 51-year-old brother. The brothers were both tall and slender and had long limbs; the younger was diagnosed as having Marfan syndrome. Both patients both showed a tendency to retarded mental development. gamma-Glutamyltranspeptidase activity was below the detection limit of 1 IU/L in both patients. Glutathionaemia and glutathionuria were evident in both brothers. The analyses of sulphydryl compounds in the plasma (and serum for certain test items) and urine indicated high concentrations of glutathione, gamma-glutamylcysteine, cysteine and cysteinylglycine. Urine amino acid analysis on an automatic analyser showed a slightly increased excretion of cystine and a large peak in the citrulline position due, at least in part, to thio-compounds.

Year-Long Validation Study and Reference Values for Urinary Amino Acids Using a Reversed-Phase HPLC Method

Reversed-phase HPLC (RP-HPLC) has become an alternative to ion-exchange chromatography for amino acid analysis in biological fluids. However, validation studies for its urine application are limited, and the corresponding reference values have not been reported extensively. We studied the long-term performance of a commercial HPLC method for urine amino acid analysis and established specific age-related reference values for urine amino acid excretion. Method performance was continuously assessed by recovery and precision studies with urine samples and controls, respectively. Healthy individuals were prospectively analyzed throughout a 5-year period. Excretion of individual amino acids, expressed as mmol/mol of creatinine, was included in six age-related groups for random urine samples (0-1 month, 1-12 months, 1-3 years, 3-8 years, 8-16 years, and >16 years) and in two groups for 24-h urine collections (8-16 years and >16 years). Over a 1-year period, CVs for retention times were <0.5% and 3.3% for within- and between-run imprecision, respectively. For amino acid concentrations, within-run CVs were 2.9-17% and between-run CVs were 7.1-46% for the same period. Amino acid recoveries were 78-122%. Reference intervals for 35 amino acids were calculated and compared with the concentrations observed in patients diagnosed with specific pathologies. A few statistically significant differences were found between the reference intervals derived using random and 24-h urine collections. Long-term reliability of the RP-HPLC method for urine amino acid analysis has been demonstrated. Representative age-related reference intervals for the RP-HPLC method in both random urine and 24-h urine collections have been established, and their feasibility for diagnosis of aminoaciduria has been shown. These intervals could serve as a guide for laboratories changing to HPLC methods.

Metabolism of l-[ guanidinooxy- 14C]canavanine in the rat

The metabolism of l-canavanine, a nonprotein amino acid with significant antitumor effects, was investigated. l-Canavanine, provided at 2.0 g/kg, was supplemented with 5 μCi of l-[ guanidinooxy- 14C]canavanine (58 μCi/μmol) and administered iv, sc, or orally to female Sprague-Dawley rats weighing approximately 200 g. 14C recovery in the urine at 24 hr was 83, 68, or 61%, respectively, of the administered dose. Another 5–8% of the 14C was expired as 14CO 2. The gastrointestinal tract contained 21% of orally administered 14C. Serum, feces, tissues, and de novo synthesized proteins only accounted for a few percent of the original dose by any administrative route. Analysis of the 14C-containing urinary metabolites revealed that [ 14C]urea accounted for 88% of the urinary radioactivity for an iv injection, 75% for sc administration, and 50% following an oral dose. By all routes of administration, [ 14C]guanidine represented 5% of the radioactivity in the urine and [ 14C]guanidinoacetic acid accounted for 2%. Serum and urine amino acid analysis showed a markedly elevated ornithine level. Basic amino acids such as histidine, lysine, and arginine were also higher in the urine. Plasma ammonia levels were determined following oral canavanine doses of 1.0, 2.0, and 4.0 g/kg. A rapid but transient elevation in plasma ammonia was observed only at the 4.0 g/kg dose. This indicates that elevated plasma ammonia is not a likely cause of canavanine toxicity at the drug concentrations used in this study.

Urine amino acid analysis by HPLC in the investigation of inborn errors of meta10545sm

Journal of Inherited Metabolic DiseaseVolume 9, Issue S2 p. 250-253 Short Communication Urine amino acid analysis by HPLC in the investigation of inborn errors of meta10545sm J. M. Leah, J. M. Leah Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorT. Palmer, T. Palmer Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorM. Griffin, M. Griffin Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorC. J. Wingad, C. J. Wingad Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorA. Briddon, A. Briddon Clinical Chemistry Department, Queen Elizabeth Hospital for Children, London, E2 8PS UKSearch for more papers by this authorV. G. Oberholzer, V. G. Oberholzer Clinical Chemistry Department, Queen Elizabeth Hospital for Children, London, E2 8PS UKSearch for more papers by this author J. M. Leah, J. M. Leah Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorT. Palmer, T. Palmer Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorM. Griffin, M. Griffin Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorC. J. Wingad, C. J. Wingad Department of Life Sciences, Trent Polytechnic, Nottingham, NG11 8NS UKSearch for more papers by this authorA. Briddon, A. Briddon Clinical Chemistry Department, Queen Elizabeth Hospital for Children, London, E2 8PS UKSearch for more papers by this authorV. G. Oberholzer, V. G. Oberholzer Clinical Chemistry Department, Queen Elizabeth Hospital for Children, London, E2 8PS UKSearch for more papers by this author First published: 01 June 1986 https://doi.org/10.1007/BF01799720Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume9, IssueS2June 1986Pages 250-253 RelatedInformation

N tau-Ribosylhistidine, a novel histidine derivative in urine of histidinemic patients. Isolation, structure, and tissue level.

On amino acid analysis of urine of histidinemic patients, an unidentified compound was eluted in a position between beta-aminoisobutyric acid and gamma-aminobutyric acid. This compound was purified to homogeneity from the urine by a combination of extraction with 80% ethanol, repeated column chromatography on Bio-Rad AG-50, and high performance liquid chromatography on a strongly cationic ion exchanger. The compound yielded free histidine on hydrolysis in an evacuated sealed tube with 0.1-6.0 M HCl at 145 degrees C for 5 h, but not at 100 degrees C for 24 h. This compound was determined to be N tau-ribosylhistidine by 1H and 13C NMR spectroscopies. The urinary content of this material in normal and histidinemic children was 17.8 +/- 13.4 (n = 10) and 126 +/- 51 (n = 14) mumol/g creatinine (mean +/- S.D.), respectively, and were closely correlated with those of urinary histidine. The renal clearance value of N tau-ribosylhistidine in humans was 96% of that of creatinine. When rats were fed on diets rich in histidine, the urinary excretion of N tau-ribosylhistidine increased greatly and was well correlated with the intake of histidine.

Serum and urine amino acid analysis in children with biliary atresia.

CHIBA, T. Serum and Urine Amino Acid Analysis in Children with Biliary Atresia. Tohoku J. exp. Med., 1983, 139 (1), 61-66 - Serum and urine amino acid levels were measured preoperatively and at varying time following successful hepatic portoenterostomy. All of the essential amino acids measured returned to normal levels during the late postoperative period over five years, whereas many of the non-essential amino acids remained in the abnormal range.

A Congenital Hair Defect: Trichoschisis With Alternating Birefringence and Low Sulfur Content

Hair from a four year old white girl with a history of brittle hair since birth did not show any previously reported defect with light microscopy. Examination with polarized light revealed regular alternating areas of birefringence and non-birefringence, suggesting a patterned abnormality of hair keratiniziation. Fractures occurred only in the non-birefringent zones. Examination of hairs from each member of the patient's family, and from normal children of the same age, did not show this pattern. Scanning electron micrographs of the defective hair shaft and fracture sites were compared with normal hair and artificially fractured hair and revealed a marked anatomic defect of the cuticle and of the fracture sites. Stress strain curve analysis of the defective hair showed a very low elongation prior to breakage, usually less than 10%. Friction coefficients between crossed hair pairs were lower than normal and correlated with the defective cuticle formation. Electron-probe x-ray microanalysis of the defective hair shaft revealed a marked deficiency of sulfur, the periodicity of which differs from that found in the normal hair. Total sulfur content of the defective hair shafts was markedly decreased. Copper content of the hair shaft was greater than the normal control. However, the copper content of the root and the root/hair shaft ratio were less than normal. Complete amino acid analysis of the patient's hair demonstrates a major decrease in the amount of cystine-cysteine. Urine amino acid analysis detected two peaks not present in normal urine. Preliminary evidence indicates that the unidentified peaks represent peptides of complex composition. Argininosuccinic acid, reported in association with trichorrhexis nodosa, was not found.