Quantification of urinary zwitterionic organic acids using weak-anion exchange chromatography with tandem MS detection.

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r2>or=0.995), accuracy (85-115%), precision (C.V.<12%), sample preparation stability (

[Megaloblastic anemia: rapid and economical study]. / Anemia megaloblástica: su estudio en forma rápida y económica.

The diagnosis of megaloblastic anaemias caused by cobalamine or folate deficiency are still difficult. The dosage of these two substances help to differenciate between both carencies, but it is not determinant of any of them and is an expensive method. Homocisteinuria (HC), methylmalonuria (MMA) and formiminoglutamic acid (FIGLU) are cheap tests which could help in the differential diagnosis, if they are used properly. We report 62 patients to whom we made these test simultaneously. All of the patients received 10 micrograms of vit B12 and after 72 hours, 1 mg/day of folic acid (for 3 days). In both cases waiting for the increase of reticulocytyes up to 150 x 10(9)/L as a form of therapeutic test of diagnosis. By this simple way we have detected 97.9% of specificity for cobalamin deficiency of the MMA test, and only 4.2% for HC. This last test had increased its specificity up to 91.6% in association with the negative FIGLU test. We have also found a high specificity (92.3%) for FIGLU due to the detection of folate deficiency, in opposition with other authors who had described it as low as 50%. We have also compared the costs of the 3 tests with the dosage of cobalamine and folate, and we have found that the formers are 11 times less expensive than the last ones.

Effect of folate supplementation on clinical chemistry and hematologic changes related to bidisomide administration in the rat.

In a chronic toxicity study in the rat, bidisomide administered as a dietary admixture produced a dose-related lowering of reticulocytes and leucocytes. Plasma alanine aminotransferase activity was increased at 300 mg/kg and decreased at 900 mg/kg. The potential mechanisms of these effects were investigated by comparing the responses in groups of male Sprague-Dawley rats receiving a control diet, or 300 or 1200 mg/kg/day bidisomide. Subsets of these groups were co-treated subcutaneously with folinic acid or with a vitamin B1, B6, B12 complex. Subsets of control and 300 mg/kg groups were maintained on a 20-25% feed restriction regimen for 3 months, to mimic the depression in body weight gain observed in animals receiving 1200 mg/kg. Body weight gains were significantly reduced at 1200 mg/kg and in all feed-restricted animals. Plasma and liver alanine aminotransferase (ALT) and plasma aspartate aminotransferase (AST) levels were also reduced at this dose level. At 300 mg/kg, plasma transaminases, glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities were increased. These changes were prevented in animals receiving folinic acid supplementation. Plasma glucose, triglycerides, and unsaturated and total iron binding capacities were decreased, while plasma iron levels tended to increase, mainly at the high dose. Vitamin supplementation prevented a decrease in reticulocyte counts at 300 mg/kg. Bidisomide increased urinary formimino-glutamic acid (FIGLU) excretion but did not affect methylmalonic acid (MMA) or taurine excretion. The effect on FIGLU at 1200 mg/kg was prevented by folinic acid co-treatment. Absolute liver weight was lowered at both dose levels and in feed-restricted animals. However, the relative liver weights were unaffected. Thymidine kinase and thymidylate synthase activity of the bone marrow cells were not altered by the bidisomide treatment. Except for the increase in plasma transaminase, GLDH and SDH levels at 300 mg/kg, changes in clinical chemistry parameters are considered to result mainly from nutritional restrictions. Changes in hematologic parameters appear to be related to the combination of decreased feed consumption (leukocytes) and decreased availability or utilization of folates (reticulocytes). This alteration, however, did not affect DNA synthesis in bone marrow. The prevention by folinic acid, but not by feed restriction, of the elevation of liver enzymes at 300 mg/kg is an intriguing, yet unexplained finding. There was no evidence that bidisomide affected B6 and B12 availability.

Cobalamin-dependent metabolism in chronic myelogenous leukemia determined by deoxyuridine suppression test and the formiminoglutamic acid and methylmalonate excretion in urine.

The cobalamin metabolism in chronic myelogenous leukemia (CML) was evaluated in 18 newly diagnosed and untreated patients by formiminoglutamic acid (FiGlu) and methyl malonic acid excretion (MMA) tests. A deoxyuridine (dU) suppression test of bone marrow cells was compared in patients with acute myelogenous leukemia (N = 5), myelodysplastic disease (N = 3), untreated pernicious anemia (N = 16), folate deficiency (N = 7), and a hospital reference group without signs of cobalamin or folate deficiency (N = 22). All had normal MMA excretion but 3 of 15 patients had increased FiGlu excretion. In vitro thymidine uptake in bone marrow cells of CML patients were lower (mean 40 fmol/106 cells) than pernicious anemia patients (115 fmol/106 cells). Methotrexate (MTX) increased the uptake in all cases. Addition of formyl-THF, methyltetrahydrofolate (methyl-THF), and pteroylglutamic acid (PGA) tended to normalize the effect of MTX. In pernicious anemia methyl-THF only decreased the uptake in combination with CN-Cbl. dU suppression values were significantly higher (6.3%) in CML than in the reference group (4.4%), but significantly lower than in pernicious anemia (41.6%) and folate deficiency (28.5%). The dU suppression values in bone marrow cells of CML patients correlated significantly with the transferrin saturation. In buffy coat cells dU suppression values were even higher (9.3%) than in bone marrow cells of the same CML patients. Addition of folate forms and CN-Cbl did not change the dU suppression values in CML, as it did in pernicious anemia. MTX increased dU suppression values significantly in all patients, but more in CML (64.5%) than in pernicious anemia (48.6%) and controls (49.8%). The MTX effect was to some extent neutralized by folate analogues with formyl-THF as the most effective followed by methyl-THF and lastly PGA. Methyl-THF also neutralized MTX in pernicious anemia, but its effect was certainly enhanced by addition of CN-Cbl. Thymidine uptake and dU suppression patterns were not significantly changed in CML after treatment with busulfan for 1 week or in accelerated phase. We concluded that signs of cobalamin or folate deficiency (apart from one patient) cannot be demonstrated in untreated CML. However, dU suppression was significantly increased and more so in circulating myeloid cells than in bone marrow. This indicates a deranged metabolism of deoxynucleotides which is independent of cobalamin and folates, and a difference between bone marrow cells and circulating cells. dU suppression is a valuable indicator of cobalamin deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)

Inter-relationships between single carbon units' metabolism and resting energy expenditure in weight-losing patients with small cell lung cancer. Effects of methionine supply and chemotherapy.

The one-carbon unit metabolism was investigated in 8 weight-losing patients with small cell carcinoma of the lung (SCLC). At diagnosis, 6 of the 8 patients had elevated formiminoglutamic acid (FIGLU) excretion after a histidine load, suggesting a lack of one-carbon units. In accordance, a significant decrease of FIGLU excretion was observed in the patients after oral administration of DL-methionine for 4 days. The elevated FIGLU excretion was positively correlated to weight loss prior to diagnosis and negatively correlated to serum albumin at time of diagnosis. After 3 months of combination chemotherapy, FIGLU excretion was reduced in all patients except 1, who had progressive disease. Despite the elevated FIGLU excretions, all patients had normal blood folate levels. The resting energy expenditure (REE) was recorded in 7 patients, and a significant, positive correlation was observed between pretreatment FIGLU excretion and REE, although the REE measured in this group of patients was within the normal range. These data demonstrate an increased demand of "active" one-carbon units in energy consumption in a group of weight-losing cancer patients. The one-carbon unit deficit was reconditioned by oral administration of the one-carbon unit donor DL-methionine.

Vitamin B12 and folate status in rats after chronic administration of ethanol and acute exposure to nitrous oxide.

The chronic administration of ethanol or brief exposure to nitrous oxide (N2O) decreases the activity of hepatic methionine synthase and disrupts normal metabolic processes that require folate and vitamin B12. This combination of drugs has clinical relevance since alcoholic patients often require surgery and receive N2O as a component of their anesthetic. To assess this clinical problem using a rodent model, rats were given a liquid ethanol diet (35% of calories as ethanol) and control rats were pair-fed a liquid diet with carbohydrate substituting for the caloric content of ethanol. After receiving liquid diets for 6 weeks, rats were exposed to 60% N2O/40% O2 for 6 hr. Urinary excretions of formic acid and formiminoglutamic acid (FIGLU) were used as indirect markers of folate status. In both the ethanol-fed and control groups, excretion of formic acid and FIGLU markedly increased the first day after N2O and returned towards background values by the second day after N2O exposure. Ethanol treatment alone decreased methionine synthase activities in liver, but not kidney or brain. Exposure to N2O further decreased methionine synthase activities, and recovery of methionine synthase activity after N2O occurred over a period of 4 days at the same rate in both the ethanol-fed and control groups. Ethanol treatment for 6 weeks combined with acute exposure to N2O did not deplete the rats of vitamin B12 in blood, liver, kidney, or brain. We conclude that in this animal model, chronic treatment with ethanol does not markedly exacerbate the disturbances in folate/vitamin B12 metabolism caused by brief exposure to N2O.

Folate deficiency and an abnormal lymphocyte deoxyuridine suppression test in monkeys.

Cebus albifrons were fed folate-deficient diets in order to assess folate status at the cellular level with the deoxyuridine suppression test. Plasma and red blood cell folates were significantly lower at 2 months, compared to control values. Hematologic signs of megaloblastic anemia occurred after 6 months, with significantly lower hematocrit, hemoglobin and red blood cell number values and increased polymorphonuclear leukocyte lobe counts. Urinary formiminoglutamic acid excretion also was elevated significantly. Whole blood lymphocyte cultures exhibited abnormal deoxyuridine suppression of [3H]-thymidine incorporation into DNA with folate deficiency. Thus this deoxyuridine suppression test can be used in isolated whole blood lymphocytes of these nonhuman primates to document folate deficiency.

Vitamin B-12 and folate function in chronic alcoholic men with peripheral neuropathy and encephalopathy.

Forty-six male alcoholics hospitalized with polyneuropathy or intellectual impairment were studied after at least 2 wk of alcohol abstention. Neurological evaluation included neurophysiological examination of the sural nerve and tibial nerve, neurophysiological examinations, and CT-scanning of the brain. Alcohol and vitamin intakes were quantified by the interview method. Vitamin B-12 and folate status included examinations of peripheral blood and bone marrow aspirate, plasma vitamin B-12, plasma and erythrocyte folate, formiminoglutamic acid excretion test (FiGlu), methylmalonic acid excretion, and deoxyuridine suppression test (dU) on phytohemagglutinin-stimulated peripheral lymphocytes. The liver function was assessed by galactose elimination capacity and plasma clearance of antipyrine. There was no hematological sign of folate or vitamin B-12 deficiency. About 8% had low plasma folate, while neither erythrocyte folate nor plasma vitamin B-12 were decreased. However, half of the patients had functional folate deficiency as determined by abnormal FiGlu or dU. Compared to the remaining patients, those with abnormal FiGlu or dU had significantly more abnormal neurophysiological tests, and lower folate intake. There was no correlation between FiGlu or dU and the quantitative liver function tests. It is concluded that 1) folate deficiency may contribute to the development of alcoholic polyneuropathy, 2) the classical parameters for folate deficiency (blood concentrations, peripheral blood, and bone marrow examinations) are not reliable in diagnosing folate deficiency and 3) functional tests like FiGlu and dU are necessary to diagnose folate deficiency in alcoholics.

Folate deficiency in rats fed diets containing free amino acids or intact proteins.

Development of folate deficiency (FD) was evaluated in weanling rats fed diets containing mixtures of free amino acids or of vitamin-free casein and gelatin as sources of dietary nitrogen. FD could be produced in 21 d with amino acid diets that promoted maximum growth rate, were completely devoid of folate and contained 1% succinylsulfathiazole. Growth retardation and blood dyscrasia associated with FD could not be demonstrated in rats fed diets containing casein and gelatin as nitrogen sources because the vitamin-free casein contained low but measurable levels of folate. The most effective protocol to produce experimental FD in rats is to feed a folate-free diet that otherwise supports maximum growth in young animals. Additional modifications such as use of methotrexate or amino acid-imbalanced or protein-deficient diets are unnecessary.

Erythrocyte formimino glutamate transferase in FIGLU aciduria.

A patient is described who presented at an early age with failure to thrive and vomiting, and had a gross excretion of formimino glutamic acid. She had normal concentration of serum folate and vitamin B12, and no haematological abnormalities, and is not mentally retarded. The Michaelis constant for erythrocyte formimino glutamate transferase was in the normal range, but the enzyme behaves differently from that from reference subjects with respect to inhibitors and activators.