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 (

Metabolic responses of folic acid and related compounds to thyroxine in rats.

This study deals with the effects of thyroidectomy and feeding thyroid powder on histidine and folic acid metabolism. Normal rats maintained on a soy protein diet, low in methionine but supplemented with vitamin B-12, oxidize approx. 10% of an injected dose of [2-14C]histidine in 3 h and excrete low levels of formiminoglutamic acid. Addition of methionine increases histidine oxidation to approx. 20%. The feeding of thyroid powder or the injection of high levels of thyroxine decreases histidine oxidation and increases formiminoglutamic acid excretion. Surgical thyroidectomy at weaning increases histidine oxidation to approx. 45% and, thus, resembles the effect of methionine in promoting histidine oxidation and decreasing formiminoglutamic acid excretion. The feeding of methionine to the thyroidectomized animal further increases histidine oxidation to 65%. The distribution of folate forms in the liver was determined by column chromatography following administration of a dose of tritiated folic acid. In the normal animal, tetrahydrofolate accounts for 38% of the total folate present. The feeding of methionine increases this to 48%, which is consistent with the observed increase in histidine metabolism. Thyroidectomy increases the percentage of tetrahydrofolate to 63% and the feeding of methionine further increases it to 68%. The percentage of tetrahydrofolate relative to total folate is in proportion to the observed rate of histidine metabolism. The action of thyroidectomy in increasing histidine oxidation may be accounted for by its effect in increasing the proportion of tetrahydrofolate.

Effect of methionine on the metabolism of formate and histidine by rats fed folate/vitamin B-12-methionine-deficient diet.

The metabolism of formate and histidine were compared in rats and in perfused livers of rats on diets deficient in vitamin B-12, methionine, and folic acid. Excretion of formate and formiminoglutamic acid, and the oxidation of [2-14C]histidine and [14C]formate to 14CO2 were measured. Liver folate levels decreased to 40% of normal on the vitamin B-12- and methionine-deficient diets but the rate of oxidation of histidine to CO2 in the whole animal decreased to 15% of normal. This indicated a reduction in the metabolic activity of the liver folates in vitamin B-12deficiency. Comparison of formate and histidine catabolism in folic acid deficiency showed that the oxidation of histine was decreased to 5% of normal but formate oxidation was decreased to only 30% of normal. This indicates that 25% of formate oxidation normally proceeds by a non-folate-dependent pathway.

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.

A liver urocanase deficiency.

Two sisters with a rare inborn error of histidine metabolism resulting from urocanase deficiency are being presented. The more common form of familial histidinemia due to histidase deficiency is excluded. The urocanase deficiency is proven by demonstrating increased excretion of metabolites of the product of the urocanase enzyme action. Further, the strongest evidence for the urocanase defect rests on the demonstration of urocanase deficiency and normal histidase activity in liver.

Skin histidase activity and urine formiminoglutamic acid (FIGLU) in patients with histidinemia found by screening newborn infants.

Skin histidase activities and urine formiminoglutamic acid (FIGLU) levels were measured in 20 patients with histidinemia, identified by Guthrie's screening method, and their family members as well as control subjects. There was a significant positive correlation between skin histidase activities and the amounts of urine FIGLU. Although the difference of skin histidase activity and the amount of urinary FIGLU was significant between any two of the three groups (i.e. controls, parents and patients; p less than 0.005), these levels ranged widely and a considerable number of the cases overlapped among groups. When a discriminant function was computed to obtain the minimum probability of misclassification between the groups using the above two parameters, a better segregation was observed. However, even though the number of misclassifications decreased, the overlapping cases were still present, especially between the parent and patient groups. It is concluded that either skin histidase activity, urine FIGLU, or both, can be used as genetic markers of the disease to a large but still limited extent.

Urinary formiminoglutamate in man. Normal values related to sex and age. Effects of low calorie intake and alcohol consumption.

A modification of the enzymic method of Tabor and Wyngarden for formiminoglutamate (FIGLU) estimation in urine is described. Alkaline degradation of FIGLU in blanks enhanced the sensitivity of the method. FIGLU excretion in 94 healthy persons indicated a sex and age dependence. Short term calorie restriction or fasting led to a decrease of FIGLU excretion while acute alcohol intoxication or chronic alcohol administration were accompanied by enhanced FIGLU excretion. It is suggested that decreased FIGLU excretion was due to histidine deficiency.

A methionine-reversible folate deficiency in rats following the acute administration of diethylnitrosamine and alpha-naphthylisothiocyanate.

Acute doses of the hepatotoxic agents diethylnitrosamine (DEN) and alpha-naphthylisothiocyanate (ANIT) to young adult male rats led to the production of a folate deficiency as determined by an elevated urinary excretion of formiminoglutamic acid (FIGLU) 2 to 4 days following administration of the compounds. High dietary levels of methionine significantly reduced the elevated levels of FIGLU produced by the 2 chemicals. Dietary folate had no significant effect on the excretion of urinary FIGLU. Although the hepatic levels of S-adenosylmethionine (SAM) were significantly increased in rats fed the high dietary levels of methionine, an acute dose of DEN did not depress the hepatic levels of SAM. The results indicate that the methyl-reversible folate deficiency caused by hepatotoxic agents is not the direct consequence of altered hepatic levels of SAM.

Fromino-glutamic acid (Figlu) excretion and abortion.

PIP: The relationship between excretion of forminoglutamic acid (figlu) in the urine and spontaneous abortion in the first and second trimesters of pregnancy was investigated in 135 nonanemic women after spontaneous abortion and curettage. The 135 were subdivided into Group A, 95 cases with no known reason for spontaneous abortion, and Group B, 40 women in whom probable abortion cause was known; Group C was control. In Group A, figlu was detected in urine of 40 (42.1%). In Group B, where abortion causes ranged from diabetes, to toxoplasmosis, to cervical incompetence, figlu was positive in only 2 (5%) cases. 3/35 controls (8.6%) were positive for figlu. The percentage of positive figlu findings in Group A was significantly higher when compared with Group B (P .001) and with Group C (P .001). In Group A, the positive figlu excretion was statistically unrelated to previous abortions (P .05), previous pregnancies (P .05), or age groups (P .05). Further, when the 40 Group B cases and the 35 Group C cases were compared with the above, there was no change in the results. If increased figlu excretion can be considered an index of folic acid deficiency, then it is possible that this deficiency may be an etiological factor in cases of spontaneous abortion.^ieng

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.

[Biochemical and hematological parameters for the estimation of folate status in man. 3. Importance of formiminoglutamic acid excretion in the urine in comparison to other measurements]. / Biochemische und hämatologische Massstäbe zur Beurteilung des Folatstatus beim Menschen. 3. Mitteilung. Bedeutung der Formiminoglutaminsäureausscheidung im Urin im Vergleich zu anderen Messgrössen.

In a study on 250 industrial workers the amount of the formiminoglutamic acid excretion in urine was measured by an enzymatic spectrophotometric method. For the estimation of the folacine status the vitamine concentration in serum and erythrozytes was determined as well as the segmentation of the neutrophilic granulocytes. As shown in previous studies these parameters indicate the degree of folate supply. A correlation between the FIGLU excretion in urine and other folate dependent parameters could not be demonstrated. Criterial to judge the folate supply are the radioimmunological determination of folacine concentration in serum and erythrocytes as well as the estimation of the segmentation of the neutrophilic granulocytes.

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.