Metabolite diagnosis of primary hyperoxaluria type 3.

BACKGROUND: Primary hyperoxaluria type 3 (PH3) is a recently described cause of childhood renal calculi. It results from mutations in the HOGA1 gene and most cases have been diagnosed after clinical ascertainment, exclusion of other genetic hyperoxalurias and mutation testing. Metabolite testing has not been widely applied but holds promise for the rapid screening and diagnosis of patients who are not specifically suspected to have PH3. CASE-DIAGNOSIS/TREATMENT: Two cases presented with renal calculi. Urine metabolite testing by tandem mass spectrometry was performed as part of the routine diagnostic work-up for this condition. Both had significantly increased levels of the PH3 urine marker 4-hydroxyglutamate and related metabolites. The diagnosis of PH3 was confirmed by the finding of bi-allelic damaging HOGA1 mutations. CONCLUSIONS: Urine screening by tandem mass spectrometry is a rapid, high-throughput test that can detect PH3 cases that may otherwise not be diagnosed.

Intraventricular Glioblastoma Multiforme in A Child with L2-Hydroxyglutaric Aciduria.

L2-hydroxyglutaric aciduria (L2-HGA) is a rare neurometabolic disease characterized by accumulation of L2-hydroxyglutarate (L2-HG), a potential oncometabolite resulting in significant lifetime risk for cerebral tumors. Herein, we present a case of intraventricular glioblastoma multiforme (GBM) in a 16-year-old child with L2-HGA who presented with rapid functional decline and persistent vomiting. The tumor was completely resected, and the patient remained well at 2-year follow-up. Clinicians should be aware of the usual insidious nature of the disease. Rapid deterioration is unusual and should raise the suspicion of tumor development. This case also illustrates the importance of surveillance neuroimaging in patients with L2-HGA. To the best of our knowledge, only 1 case of GBM has been reported and it was sited in the temporal lobe, unlike the unusual intraventricular location in our case.

Urinary glutamine/glutamate ratio as a potential biomarker of pediatric chronic intestinal pseudo-obstruction.

Chronic intestinal pseudo-obstruction (CIPO) is a rare intestinal motility disorder with significant morbidity and mortality in pediatric patients. The diagnosis of CIPO is difficult, because it is clinically based on the symptoms and signs of bowel obstruction which are similar to the clinical manifestations of other gastrointestinal diseases like short bowel syndrome (SBS). Therefore, it is desirable to identify and establish new laboratory diagnostic markers for CIPO that are reliable and easily accessible. In our study we have identified the ratio of the urinary glutamine and glutamic acid as a promising biomarker for distinguishing suspected CIPO cases and simple SBS cases. The area under ROC curve was 0.83, at cutoff value = 7.04 with sensitivity of 65% and specificity of 92%.

Child Neurology: cognitive delay in a 7-year-old girl.

Organic acidurias are an important group of inherited metabolic disorders that affect the intermediary metabolic pathways of carbohydrate, amino acid, and fatty acid oxidation, leading to the accumulation of organic acids.(1) The 2-hydroxyglutaric acidurias are rare neurometabolic disorders characterized by developmental delay with or without other neurologic dysfunction. Three different subtypes have been described: d-2-hydroxyglutaric aciduria, l-2-hydroxyglutaric aciduria, and combined d-l-2-hydroxyglutaric aciduria. We describe the case of a child presenting with developmental delay who was found to have the classical biochemical, imaging, and genetic features of l-2-hydroxyglutaric aciduria.

Folate catabolites in spot urine as non-invasive biomarkers of folate status during habitual intake and folic acid supplementation.

BACKGROUND: Folate status, as reflected by red blood cell (RCF) and plasma folates (PF), is related to health and disease risk. Folate degradation products para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (apABG) in 24 hour urine have recently been shown to correlate with blood folate. AIM: Since blood sampling and collection of 24 hour urine are cumbersome, we investigated whether the determination of urinary folate catabolites in fasted spot urine is a suitable non-invasive biomarker for folate status in subjects before and during folic acid supplementation. STUDY DESIGN AND METHODS: Immediate effects of oral folic acid bolus intake on urinary folate catabolites were assessed in a short-term pre-study. In the main study we included 53 healthy men. Of these, 29 were selected for a 12 week folic acid supplementation (400 µg). Blood, 24 hour and spot urine were collected at baseline and after 6 and 12 weeks and PF, RCF, urinary apABG and pABG were determined. RESULTS: Intake of a 400 µg folic acid bolus resulted in immediate increase of urinary catabolites. In the main study pABG and apABG concentrations in spot urine correlated well with their excretion in 24 hour urine. In healthy men consuming habitual diet, pABG showed closer correlation with PF (rs = 0.676) and RCF (rs = 0.649) than apABG (rs = 0.264, ns and 0.543). Supplementation led to significantly increased folate in plasma and red cells as well as elevated urinary folate catabolites, while only pABG correlated significantly with PF (rs = 0.574) after 12 weeks. CONCLUSION: Quantification of folate catabolites in fasted spot urine seems suitable as a non-invasive alternative to blood or 24 hour urine analysis for evaluation of folate status in populations consuming habitual diet. In non-steady-state conditions (folic acid supplementation) correlations between folate marker (RCF, PF, urinary catabolites) decrease due to differing kinetics.

Quantification of isotope-labeled and unlabeled folates and folate catabolites in urine samples by stable isotope dilution assay.

Dual-label stable isotope dilution assays for the simultaneous quantification of isotopologic folates in clinical samples offer the perspective for differentiating between unlabeled folates from endogenous body pools and administered [13C5]-labeled folates from a test dose when performing bioavailability trials. In contrast to intact folates, this methodology could hitherto not be applied to the quantification of the folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate. In this study, [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate were synthesized. The synthesis of the [2H4]-labeled compounds started at unlabeled p-aminobenzoic acid. For the formation of p-acetamidobenzoyl glutamate, p-aminobenzoyl glutamate was acetylated. The new substances were applied successfully in stable isotope dilution assays for the simultaneous quantification of the [13C5]-labeled and unlabeled folate catabolites, p-aminobenzoyl glutamate and p-acetamidobenzoyl glutamate, along with the predominant folate vitamers in urine. The assays were based on clean-up by strong anion exchange followed by liquid chromatography-tandem mass spectrometry detection. Assay sensitivity was sufficient to detect the folate catabolites in physiologic concentrations. The limit of detection was below 0.4 and 0.3 nmol/100 g for p-aminobenzoyl glutamate isotopologues and p-acetamidobenzoyl glutamate isotopologues in urine, respectively. The successful synthesis of [2H4]-p-aminobenzoyl glutamate, [2H4]-p-acetamidobenzoyl glutamate, and unlabeled p-acetamidobenzoyl glutamate and the implementation of these substances in stable isotope dilution assays allows dual-label designs that provide a more detailed insight into human folate metabolism.

A randomized targeted amino acid therapy with behaviourally at-risk adopted children.

BACKGROUND: Increasing numbers of children are at-risk for behavioural and emotional disorders, a phenomenon contributing to increased use of pharmacological interventions for paediatric clients. Adverse side effects and other risks associated with pharmacological approaches have helped fuel interest in nutritional interventions for behaviourally at-risk children. METHODS: The current randomized clinical trial evaluates the efficacy of a neurochemical intervention involving the glutamine and glutamate analogue L-theanine and 5-hydroxytryptophan, the precursor for serotonin, with children adopted from traumatic backgrounds. RESULTS: Results include significant increases in urinary levels of the biomarkers for serotonin and gamma-aminobutyric acid, coupled with significant decreases in parent reports of the children's behaviour problems. CONCLUSIONS: While further research is needed, these initial findings are encouraging and are consistent with a growing number of studies indicating the efficacy of nutritional approaches to help behaviourally at-risk children.

L-2-hydroxyglutaric aciduria: report of four Turkish adult patients.

BACKGROUND: l-2-Hydroxyglutaric aciduria is a rare progressive neurometabolic disorder of childhood inherited as an autosomal recessive trait. Urine organic-acid screening is necessary for its diagnosis. Although it is a disorder of childhood, recently adult cases have been reported. CASES: Here we report 4 adult patients in whom diagnoses were established in adulthood. These patients had some interesting features. First, their diagnoses were delayed until adulthood because of mild clinical symptoms. In such cases, the typical MRI findings are the best diagnostic clue for l-2-Hydroxyglutaric aciduria. Second, there was a correlation between the severity of the clinical course and the extent of MRI findings. The cerebral white-matter lesions were diffuse and confluent on the MRI of 3 of the 4 patients, who also experienced a rapidly progressive clinical decline. Third, there were different clinical presentations even within the same family. CONCLUSIONS: For the evaluation of patients with symptoms referable to cerebellar, pyramidal, extrapyramidal, or cognitive impairment as well as seizures associated with subcortical white-matter and symmetrical dentate nuclei and basal ganglia involvement on MRI, urine organic acid analysis should be included in the evaluation, regardless of patient's age.

Quantitation of folates and their catabolites in blood plasma, erythrocytes, and urine by stable isotope dilution assays.

New stable isotope dilution assays were developed for the simultaneous quantitation of the folates 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, 10-formylfolic acid, and folic acid as well as for their catabolites para-aminobenzoylglutamate (pABG) and acetyl-para-aminobenzoylglutamate (ApABG) in clinical samples. The methods were based on cleanup by strong anion exchange followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. Deuterated analogues of the folates and [(13)C(5)]-labeled isotopologues of the catabolites were applied as the internal standards in stable isotope dilution assays. Extraction in 4-morpholineethanesulfonic acid (MES) buffer at pH 5.0 ensured the optimum stability of folates and, in combination with solid-phase extraction (SPE) based on strong anion exchange, resulted in higher recoveries compared with other combinations of extraction buffers and SPE. The method was sensitive enough to detect pABG in plasma generally and unmetabolized folic acid in the plasma of a volunteer after oral dosage of an aqueous folic acid solution. The sum of folate catabolites increased by a factor of 2 in the urine of the latter volunteer, compared with that resulting when only water was dosed.

Glutaric aciduria type II [corrected] and brain tumors: a case report and review of the literature.

Heritable diseases associated with childhood tumors are sometimes defined as a probable etiologic factor or a coincidence. First of all, we must know the actual number of patients. Herein a case with medulloblastoma associated with glutaric aciduria type II [corrected] is reported for this purpose. A 5-year-old boy was admitted with nausea, vomiting, and lethargy. In medical history, consanguinity and siblings with mental-motor retardation and epilepsy are remarkable. Growth retardation, macrocephaly, lethargy, tremor, bilateral nistagmus, and papilledema were prominent features in physical examination. Noncontrast computed tomography of the brain showed a hyper dense mass in the cerebellar vermis. Gross total resection was made and the histopathology of the tumor was medulloblastoma. Besides medical history and physical findings, radiologic white matter changes in the subcortical, periventricular regions, bilateral basal ganglia, and caudate nuclei in magnetic resonance images other than tumor led us to investigate the child for glutaric aciduria type II [corrected]. The level of the 2-OH glutaric acid was determined as being 12-fold high in the urine. Chemo-radiotherapy was performed after surgery. Our case was the third patient with medulloblastoma in the literature and is still alive with no evidence of the disease 19 months after the initial diagnosis.

Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry.

OBJECTIVE: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 microL) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. RESULTS: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 microL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 micromol/L for urines. Imprecision for spiked serum samples (n=10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n=10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/L and serum folate of 35.3+/-5.8 nmol/L. Serum from patients being tested for PTH (n=11) had serum folate levels of 27.0+/-10.4 nmol/L with total serum catabolites of 20.4+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n=19) was 581.8+/-368.4 nmol/L. CONCLUSION: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.

Phase I and pharmacokinetic study of pemetrexed administered every 3 weeks to advanced cancer patients with normal and impaired renal function.

PURPOSE: This phase I study was conducted to determine the toxicities, pharmacokinetics, and recommended doses of pemetrexed in cancer patients with normal and impaired renal function. PATIENTS AND METHODS: Patients received a 10-minute infusion of 150 to 600 mg/m2 of pemetrexed every 3 weeks. Patients were stratified for independent dose escalation by measured glomerular filtration rate (GFR) into four cohorts ranging from > or = 80 to less than 20 mL/min. Pemetrexed plasma and urine pharmacokinetics were evaluated for the first cycle. Patients enrolled after December 1999 were supplemented with oral folic acid and intramuscular vitamin B12. RESULTS: Forty-seven patients were treated with 167 cycles of pemetrexed. Hematologic dose-limiting toxicities occurred in vitamin-supplemented patients (two; 15%) and non-supplemented patients (six; 18%), and included febrile neutropenia (four patients) and grade 4 thrombocytopenia (two patients). Nonhematologic toxicities included fatigue, diarrhea, and nausea, and did not correlate with renal function. Accrual was discontinued in patients with GFR less than 30 mL/min after one patient with a GFR of 19 mL/min died as a result of treatment-related toxicities. Pemetrexed plasma clearance positively correlated with GFR (r2 = 0.736), resulting in increased drug exposures in patients with impaired renal function. With vitamin supplementation, pemetrexed 600 mg/m2 was tolerated by patients with a GFR > or = 80 mL/min, whereas patients with a GFR of 40 to 79 mL/min tolerated a dose of 500 mg/m2. CONCLUSION: Pemetrexed was well tolerated at doses of 500 mg/m2 with vitamin supplementation in patients with GFR > or = 40 mL/min. Additional studies are needed to define appropriate dosing for renally impaired patients receiving higher dose pemetrexed with vitamin supplementation.

Pharmacokinetics of theanine enantiomers in rats.

Theanine, first discovered in tea, is a chiral nonproteinic amino acid that has been reported to have cardiovascular, neurological, and oncological effects. It is being considered as a therapeutic/medicinal agent and additive in consumer products. The present study evaluated the pharmacokinetics of D-theanine, L-theanine, and D,L-theanine in plasma and urine using LC-ESI/MS in rats after oral (p.o.) and intraperitoneal (i.p.) administration. Oral administration data indicated that gut absorption of d-theanine was far less than that of L-theanine. However, after i.p. administration, plasma theanine concentrations of L- and D-theanine were similar. This indicated that D- and L-theanine may exhibit a competitive effect with respect to intestinal absorption. Regardless of the route of administration, p.o. or i.p., the presence of the other enantiomer always decreased theanine plasma concentrations, indicating D,L-theanine competition with respect to urinary reabsorption. Data on urinary concentrations of D-theanine suggested that the D-isomer may be eliminated with minimal metabolism. L-Theanine appeared to be preferentially reabsorbed and metabolized by the kidney while D-theanine was preferentially excreted. Clearly, the bioequivalencies of D,L-theanine and its enantiomers were found to be quite different from one another. Consequently, the efficacy of commercial theanine products containing D-theanine, L-theanine, or D,L-theanine may be quite different.

Folate catabolite excretion is responsive to changes in dietary folate intake in elderly women.

BACKGROUND: The major route of folate turnover is by catabolic cleavage of the C9-N10 bond producing p-aminobenzoylglutamate (pABG) and its primary excretory form, p-acetamidobenzoylglutamate (ApABG). We hypothesize that total pABG (ApABG + pABG) excretion parallels both the mass of body folate pools from which these catabolites originate and the folate-status indicators. OBJECTIVE: The objective was to determine whether urinary folate catabolite excretion reflects body pool size and parallels the static and functional measures of folate status. DESIGN: Urinary folate catabolite excretion was measured in women (aged 60-85 y) consuming controlled amounts of folate for 14 wk. A low-folate diet (120 microg/d) was consumed (n = 33) for 7 wk, and then subjects were randomly assigned to consume either 200 (n = 14) or 400 (n = 16) microg folate/d. Urinary pABG and ApABG concentrations were measured by HPLC at 0, 7, and 14 wk. RESULTS: Urinary excretion of total pABG was significantly lower (P = 0.001) after depletion (73.9 +/- 4.7 nmol/d) than at baseline (115 +/- 12.7 nmol/d). This rate of decline (approximately 0.7% per day) is consistent with the kinetically measured rate of turnover of total body folate at moderate folate intakes. The average percentage increase in total pABG in response to folate repletion with 400 microg/d (75%) was significant (P = 0.02). Folate catabolite excretion was significantly (P = 0.0001) associated with serum and red blood cell folate, plasma homocysteine, and DNA hypomethylation after depletion and with serum folate (P = 0.001) and plasma homocysteine (P = 0.0002) after repletion with 400 microg folate/d. CONCLUSIONS: Total urinary pABG excretion reflects total body folate pool size and is a long-term indicator that parallels functional measures of folate status.

Consumption of the folate breakdown product para-aminobenzoylglutamate contributes minimally to urinary folate catabolite excretion in humans: investigation using [(13)C(5)]para-aminobenzoylglutamate.

Folate catabolism represents the major route of folate turnover in humans and involves cleavage of the C9-N10 bond producing a pterin and para-aminobenzoylglutamate (pABG). Thus, the quantitation of pABG and its acetylated more predominant counterpart para-acetamidobenzolyglutamate (apABG) may be useful in assessing folate status and requirements. However, until the in vivo fate of dietary pABG is understood, studies using pABG excretion parameters can not be fully interpreted. As part of a larger study, an oral dose (376 nmol or 100 micro g) of [(13)C(5)]pABG in 40 mL apple juice was ingested by pregnant women (2nd trimester, n = 2) and nonpregnant controls (n = 2) consuming controlled total folate intakes of 450 or 850 micro g/d. Urine collections (24 h) were obtained over the next 4 d and gas chromatography-mass spectrometry was used to measure urinary [(13)C(5)]pABG, [(13)C(5)]apABG and [(13)C(5)]folate. Of the 376 nmol [(13)C(5)]pABG administered, only 17.5 +/- 6.4 nmol; mean +/- SEM) or 4.6 +/- 1.7% of the dose was accounted for in the urine. Most of the excreted [(13)C(5)]pABG, in acetamido form (15.1 +/- 5.3 nmol), was excreted the day after the dose. No urinary [(13)C(5)]folate was detected. Folate intake did not seem to influence the urinary excretion of total pABG derived from oral pABG, whereas pregnancy may lessen total pABG excretion derived from oral pABG. Overall, these results suggest that the contribution of dietary pABG to the urinary excretion of pABG and apABG is small.

L-2-hydroxyglutaric aciduria in two siblings.

Two Pakistani siblings with L-2-hydroxyglutaric aciduria are reported herein. A 6-year-old male and a 2-year-old female, born to consanguineous parents, had chronic slowly progressive neurodegenerative disorder with insidious onset after infancy. Mental regression and seizures were evident in both patients, whereas cerebellar dysfunction was the main motor handicap in the male and pyramidal symptoms were prominent in the female. Magnetic resonance imaging revealed bilateral symmetrical abnormal signal in the subcortical white matter, internal and external capsules, basal ganglia, and dentate nuclei. The underlying metabolic defect, which is likely inherited in an autosomal recessive mode, remains unknown in this disorder.

Bioactivation of benzylamine to reactive intermediates in rodents: formation of glutathione, glutamate, and peptide conjugates.

The in vivo and in vitro disposition of benzylamine was investigated in rats. Benzylamine was metabolized to only a small extent by rat liver subcellular fractions. In contrast, it was extensively metabolized in vivo in rats. In vivo studies performed with stable isotope-labeled benzylamine enabled rapid mass spectrometric identification of metabolites present in rat bile and urine. The major metabolite of benzylamine was the hippuric acid formed by glycine conjugation of benzoic acid. LC/MS analysis of bile and urine obtained from rats dosed with 1:1 equimolar mixture of either d(0):d(7)- or d(0):d(2)-benzylamine showed the presence of several glutathione adducts in addition to the hippuric acid metabolite. The presence of various glutathione adducts indicated that benzylamine was metabolized to a number of reactive intermediates. Various metabolic pathways, including those independent of P450, were found to produce these intermediates. A previously undocumented pathway included the formation of a new carbon-nitrogen bond that led to a potentially reactive intermediate, Ar-CH(2)-NH(CO)-X, capable of interacting with various nucleophiles. The origin of this reactive intermediate is postulated to occur via the formation of either a formamide or carbamic acid metabolites. Metabolites which were produced by the reaction of this intermediate, Ar-CH(2)-NH(CO)-X with nucleophiles included S-[benzylcarbamoyl] glutathione, N-acetyl-S-[benzylcarbamoyl]cysteine, S-[benzylcarbamoyl] cysteinylglycine, S-[benzylcarbamoyl] cysteinylglutamate, N-[benzylcarbamoyl] glutamate, and an oxidized glutathione adduct. Bioactivation of amines via this pathway has not been previously described. The oxidative deamination of benzylamine yielding the benzaldehyde was demonstrated to be a precursor to the hippuric acid metabolite and S-benzyl-L-glutathione. The formation of the S-benzyl-L-glutathione conjugate showed that a net displacement of amine from benzylamine had taken place with a subsequent addition of glutathione at the benzylic position. In addition to these novel pathways, a number of other glutathione-derived adducts formed as a result of epoxide formation was characterized. It was demonstrated that benzylamine was converted by rat P450 2A1 and 2E1 to benzamide that was rapidly metabolized to an epoxide. Mechanisms are proposed for the formation of various GSH adducts of benzylamine.

Kinetics of folate turnover in pregnant women (second trimester) and nonpregnant controls during folic acid supplementation: stable-isotopic labeling of plasma folate, urinary folate and folate catabolites shows subtle effects of pregnancy on turnover of folate pools.

To investigate the effects of pregnancy on folate metabolism, we conducted an 84-d study in second-trimester (gestational wk 14-25) pregnant women (n = 6) and nonpregnant controls (n = 6) with stable-isotopic tracer methods. All subjects were fed a diet containing approximately 272 nmol/d (120 microg/d) folate from food, along with supplemental folic acid that contained 15% [3',5'-(2)H(2)] folic acid ([(2)H(2)]folic acid) during d 1--41 and that was unlabeled during d 42--84 to yield a constant total folate intake of 1.02 or 1.93 micromol/d (450 or 850 microg/d). Isotopic enrichment of plasma folate, urinary folate and the urinary folate catabolites para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG) was determined at intervals throughout the study. The labeling of pABG and ApABG reflected that of tissue folate pools from which the catabolites originate. After the intake of labeled folic acid was terminated on d 41, labeling of urinary folate exhibited a biphasic exponential decline with distinct fast and slow components. In contrast, during d 42--84, the enrichment of urinary pABG and ApABG exhibited primarily monophasic exponential decline, and plasma folate underwent little decline of labeling during this period. Pregnant women and controls did not differ in estimates of body folate pool size and most aspects of the excretion of labeled urinary folate and catabolites, rates of decline of excretion, and areas under the curves for folate and catabolite excretion. Pregnant women, however, tended to have a slower rate of decline of pABG than ApABG and higher enrichment at d 42 of ApABG and pABG. These data support and extend our previous findings indicating that pregnancy (gestational wk 14--26) causes subtle changes in folate metabolism but does not elicit substantial increases in the rate or extent of folate turnover at these moderately high folate intakes.

Highly sensitive analysis of the antifolate pemetrexed sodium, a new cancer agent, in human plasma and urine by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of pemetrexed (LY231514, ALIMTA) in human urine and plasma. Plasma samples were spiked with the internal standard lometrexol and extracted using Certify II columns. Pemetrexed was assayed in diluted urine by an external calibration method. A C8 column was used for the separation of analytes with a mobile phase composed of sodium formate buffer and acetonitrile. Between- and within-day precision and accuracy were acceptable down to the limit of quantitation of 5 ng/ml in plasma. This method was used successfully for an investigation of the disposition of pemetrexed in patients receiving 500 mg/m2 as a 10-min infusion.

Urinary excretion of folate catabolites responds to changes in folate intake more slowly than plasma folate and homocysteine concentrations and lymphocyte DNA methylation in postmenopausal women.

Folate turnover involves urinary excretion, fecal excretion, and catabolism that involves cleavage of the C9-N10 bond to yield pterins and para-aminobenzoylglutamate (pABG). Little is known about the relationship between the function of folate pools and their rates of catabolism. We report here an investigation of excretion of urinary pABG and its primary excretory form, para-acetamidobenzoylglutamate (ApABG) in samples collected during a previously published study of postmenopausal women. Ten women (49-63 y) were fed a low folate diet (56 microg/d) supplemented with folic acid to yield total folate intakes of 195 microg/d (d 1-5), 56 microg/d (d 6-41), 111 microg/d (d 42-69), 286 microg/d (d 70-80) and 516 microg/d (d 81-91). This caused changes in plasma folate, plasma homocysteine and global methylation of lymphocyte DNA. For each subject, a 7-d pooled urine sample was collected over d 1-7, 36-42, 64-70 and 85-91. ApABG constituted >85% of total catabolite excretion, and folate intake did not significantly influence ApABG or pABG excretion. The molar ratio of total catabolite excretion/folate intake varied significantly, with ratios of 1.0 +/- 0.17 (d 1-7), 3.0 +/- 0.55 (d 36-42), 1.1 +/- 0.18 (d 64-70) and 0. 33 +/- 0.054 (d 85-91). These observations indicate that the rate of folate catabolite excretion is related mainly to masses of slow-turnover folate pools governed by long-term folate intake. Folate pools functioning in some forms of methyl group metabolism respond to dietary changes in folate intake much more rapidly.