Folate sufficient subjects do not accumulate additional folates during supplementation.

In a double-blinded placebo-controlled trial of folic acid supplementation in 82 alcoholic subjects, it was found that whole blood folate levels, determined by a mass spectrometric method, do not increase in subjects whose baseline folate levels are above the third quartile (folate sufficiency). Since a state of folate sufficiency can now be identified, a recommended daily allowance (RDA) for folate can be determined using objective means.

Glutathione oxidation in real time by thermospray liquid chromatography-mass spectrometry.

The oxidation and reduction of glutathione and oxidized glutathione were studied in real time by liquid chromatography-mass spectrometry during exposure to hydrogen peroxide and mercaptoethanol. By mass spectrometry mixed disulfides and both reversible and irreversible oxidations of sulfur to higher states (sulfinic and sulfonic acids) were directly observed during exposure to hydrogen peroxide. The irreversible oxidation of glutathione to glutathione sulfonic acid could be detected after 30 min exposure of glutathione to 40 mM H2O2 at 20 degrees C. A peak consistent with glutathione-sulfinic acid was transiently present, suggesting this compound behaved as an oxygen consuming antioxidant. Liquid chromatography-mass spectrometry appears to be an excellent method to study oxidation and reductions of sulfur containing peptides and amino acids.

Splenosis presenting as an ulcerated gastric mass: endoscopic and endoscopic ultrasonographic imaging.

A case of an ulcerated gastric wall mass ultimately found to be splenosis is presented in which the index patient had endoscopic and endoscopic ultrasonographic evaluation prior to resection. Although no visual features identified this mass as a splenic implant preoperatively, the lesion appeared to be atypical for leiomyoma, which led to surgical intervention. The role of endoscopic ultrasonography in assessing isolated gastric masses is discussed.

Normal digestive physiology and the evaluation of digestive function.

Normal digestion is a complex and coordinated process that breaks food into constitutive molecules, some of which are absorbed and others passed through the body as waste. Many of the conditions associated with wasting syndromes alter normal digestive function and can lead to or exacerbate malnutrition. Digestive function can be defined by applying the principles of normal physiology. Our laboratory has developed a relatively comprehensive stable isotope method to assess function, which shows promise as a screening tool in evaluating patients with wasting syndromes. This and other tests of digestive function can be used to define the abnormalities associated with wasting and to direct or optimize antiwasting therapy.

Physiologic response to a protein, carbohydrate, fat meal in patients with human immunodeficiency virus who underwent small intestinal enteropathy as characterized by a kinetic model of D-xylose absorption.

PURPOSE: Small intestinal human immunodeficiency virus enteropathy is characterized by profound absorptive dysfunction unrelated to histology or pathogens. Frequently an attempt is made to compensate for this intestinal failure by supplementing nutrient intake with nourishing liquid meals. It is not known how the diminished absorptive function in these patients will respond to this intake. With the use of a D-xylose kinetic model of absorption, we determined the absorptive response of patients with small intestinal enteropathy to an isotonic liquid feeding. METHODS: Seven male patients with acquired immunodeficiency syndrome (AIDS), diarrhea, weight loss, and no detectable pathogens (stool studies and duodenal biopsy) were enrolled. After an overnight fast, the patients were studied on three separate days. On day 1, the patients received 15 g oral D-xylose. On day 2, 10 g i.v. D-xylose was given. On day 3, 15 g oral D-xylose was again given along with 250 mL of a liquid polymeric isotonic diet. Serum and urine collections were obtained to calculate the kinetic rate constants and extent of D-xylose absorption. RESULTS: Mean values for the rate constant for absorption of D-xylose, Ka, (0.26/h; N > 0.65) and the rate constant for nonabsorptive loss, K0' (2.47/h; N < 0.353) were very abnormal before the meal. Mean K0 improved (decreased to 0.66), but Ka and bioavailability, F, did not have a statistically significant change after the meal. The improvement in mean K0 with the meal was much more pronounced in the five subjects with high K0 values before the meal (without meal 3.22: with meal 0.67; p < .05). CONCLUSIONS: (1) An isotonic liquid polymeric diet leads to less nonabsorptive loss of D-xylose, but does not affect the extent of D-xylose absorption in this group as a whole. This is probably due to the meal slowing gastric emptying. (2) Improvement in nonabsorptive loss with a meal is most pronounced when there is excessive nonabsorptive loss, K0, without a meal. (3) Improvement in nonabsorptive losses with a meal might predict which patients will benefit from antimotility agents and continued feedings vs those requiring i.v. hyperalimentation.

Determination of p-hydroxyphenylpyruvate, p-hydroxyphenyllactate and tyrosine in normal human plasma by gas chromatography-mass spectrometry isotope-dilution assay.

The synthesis and purification of [13C2]p-hydroxyphenyllactic acid from [13C2]p-hydroxyphenylpyruvic acid, the characterization of tert.-butyldimethylsilyl-derivatized tyrosine, p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid, and an isotope-dilution assay for these substances in normal human plasma using gas chromatography-mass spectrometry (GC-MS) are described. Using this method plasma p-hydroxyphenylpyruvate, p-hydroxyphenyllactate and tyrosine levels of 68 +/- 42 ng/ml, 118 +/- 45 ng/ml and 16.6 +/- 6.3 micrograms/ml, respectively, were found in 9 normal adults. Isotope-dilution assays are sensitive enough to determine tyrosine, p-hydroxyphenylpyruvate and p-hydroxyphenyllactate content in normal subjects, and may be useful for studying disorders of tyrosine metabolism, including inborn errors of metabolism, liver disease and ascorbic acid deficiencies.

Gas chromatographic/mass spectrometric measurement of ascorbic acid and analysis of ascorbic acid degradation in solution.

L-Ascorbic acid, DHA, and the oxidized products derived from AA can be accurately measured using GC/MS. Owing to the complex nature of the reactions through which AA proceeds, we believe that GC/MS is currently the procedure of choice in making AA-related measurements. The methods described are useful in defining reactions involving AA. The methods may indicate in vivo oxidative injury and may allow the use of AA-derived products to determine if antioxidant modulations are effective.

A noninvasive stable-isotope method to simultaneously assess pancreatic exocrine function and small bowel absorption.

OBJECTIVE: To determine if a single-step noninvasive stable isotope method of assessing digestive function could separate normal subjects from subjects with pancreatic insufficiency (maldigestion) or small bowel dysfunction (malabsorption) and to see if subjects with maldigestion could be simultaneously separated from subjects with malabsorption. METHODS: Forty (40) normal volunteers, 18 adults with cystic fibrosis and four adults with celiac sprue, ingested a liquid test meal along with bentiromide, [13C6]PABA, and xylose (PABAX test). Serum was collected at 1 h and analyzed for PABA, [13C6]PABA, and xylose by stable isotope dilution methods using gas chromatography mass spectrometry. RESULTS: All subjects with cystic fibrosis had abnormal pancreatic function test results, whereas three of four adults with sprue had normal values of pancreatic function. All subjects with sprue had abnormal small bowel absorption tests, whereas all adults with cystic fibrosis had apparently normal intestinal function. CONCLUSION: The one-step, 1-h PABAX test can reliably separate normal subjects from those with either maldigestion or malabsorption and can also separate subjects with maldigestion from those with malabsorption.

Quantitation of red blood cell folates by stable isotope dilution gas chromatography-mass spectrometry utilizing a folate internal standard.

We report a new gas chromatography-mass spectrometry (GC-MS) method of measurement of red blood cell folates utilizing a stable isotope-labeled bacterial synthesized folate internal standard. The GC-MS method exploits the fact that the common feature of all folate molecules is a p-aminobenzoic acid moiety sandwiched between a pteridine ring and a polyglutamate chain of varying length. In this method, red blood cell folates together with a folate internal standard are specifically purified using bovine folate binding protein and the folates are subsequently chemically cleaved to p-aminobenzoic acid, pteridines, and glutamic acids. Since all six carbon atoms of the benzene ring in the p-aminobenzoic acid moiety of the folate internal standard are labeled with [13C], it is possible to use selected ion monitoring and stable isotope dilution GC-MS to quantitate folates. The method appears to be sensitive, specific, and accurate. The method has been applied to generate a reference range of red blood cell folates based on assay of 25 normal individuals.

Serum xylose analysis by gas chromatography/mass spectrometry.

A gas chromatography/mass spectrometric (GC/MS) isotope dilution assay for xylose was developed using tertbutyldimethylsilyl-derivatized xylose and [13C]1xylose, and applied to human serum samples. A calibration curve in serum using this assay showed < 3% variation (< 10 mg/L) for any given point. The correlation coefficient for xylose measurements made on 27 sera between a colorimetric method performed by a national commercial reference laboratory and the GC/MS method developed here was .952. However, xylose determinations of 10 of 27 samples differed by > 10% (up to 150 mg/L) when colorimetric values were compared to GC/MS. Two of these samples had borderline-low xylose values by GC/MS, but were well within the normal range by colorimetric analysis. gas chromatography/mass spectrometric isotope dilution assay appears to be an accurate method to measure xylose in serum. These data also suggest that further prospective studies comparing GC/MS to colorimetric methods are indicated for subjects undergoing oral xylose testing.

Are neuropsychiatric manifestations of folate, cobalamin and pyridoxine deficiency mediated through imbalances in excitatory sulfur amino acids?

Folate, cobalamin and pyridoxine deficiency are associated with psychiatric or neurological symptomatology. Disturbances in sulfur amino acid metabolism leading to accumulation of homocysteine occurs in all three conditions as the metabolism of homocysteine depends on enzymes requiring these vitamins as cofactors. Oxidation products of homocysteine (homocysteine sulfinic acid and homocysteic acid) and cysteine (cysteine sulfinic acid and cysteic acid) are excitatory sulfur amino acids and may act as excitatory neurotransmitters, whereas taurine and hypotaurine (decarboxylation products of cysteic acid and cysteine sulfinic acid) may act as inhibitory transmitters. Homocysteic acid and cysteine sulfinic acid have been considered as endogenous ligands for the N-methyl-D-aspartate (NMDA) type of glutamate receptors. The profile of these sulfur amino acid neurotransmitters could be altered in a similar fashion in states of decreased availability of folate, cobalamin or pyridoxine. It is proposed that the mechanism of neuropsychiatric manifestations in all three conditions result from a combination of two insults to homocysteine catabolism in the brain.

Measurement of excitatory sulfur amino acids, cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid, and homocysteic acid in serum by stable isotope dilution gas chromatography-mass spectrometry and selected ion monitoring.

Oxidized sulfur-containing amino acids are recognized as agonists of excitatory amino acid receptors in the mammalian nervous system. Homologues of glutamic acid (homocysteine sulfinic acid and homocysteic acid) and aspartic acid (cysteine sulfinic acid and cysteic acid) have been shown to be agonistic to N-methyl-D-aspartate receptors in animal brain and have been demonstrated in brain tissue. Considerable evidence exists for the role of homocysteic acid and cysteine sulfinic acid as endogenous ligands for excitatory amino acid receptors. We report, for the first time, the quantitation of these compounds in normal human serum, by a newly developed gas chromatography-mass spectrometry method that employs stable isotope-dilution selected ion monitoring using internal standards prepared in our laboratory. We also report new methods of synthesis of stable isotope-labeled internal standards used in measuring cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid, and homocysteic acid.

Ascorbate and dehydroascorbate measurements in aqueous solutions and plasma determined by gas chromatography-mass spectrometry.

The tert-butyldlmethylsllyl derivatives of ascorbic acid and dehydroascorbic acid were characterized by gas chromatography-mass spectrometry, and an isotope dilution assay for ascorbate and dehydroascorbate was developed using [13C6]ascorbic acid and [13C6]- and [6,6-2H2]dehydroascorbate. This assay was used to monitor ascorbic acid loss and the resulting rise of dehydroascorbic acid in aqueous solutions and plasma. Ascorbic acid was shown to rapidly decompose in aqueous solutions containing transition metal ions or when exposed to oxygen. Ethylenedlaminetetraacetic acid chelation did not prevent ascorbic acid degradation in aqueous solution, and ascorbate in ethylenedlaminetetraacetic acid chelated plasma was converted to dehydroascorbate on freezing. Gas chromatography-mass spectrometry appears to be a satisfactory method for determining the ascorbate and dehydroascorbate content of solutions including human blood plasma, whether or not there is ongoing oxidation of ascorbate in those solutions.

The conversion of the human membrane-associated folate binding protein (folate receptor) to the soluble folate binding protein by a membrane-associated metalloprotease.

The membrane-associated (M-FBP) and soluble (S-FBP) forms of human folate binding proteins (FBP) have been well characterized. Although related in a precursor-product manner, the mechanism of conversion and the basis for differences between M-FBP and S-FBP are not known. The conversion of M-FBP to S-FBP in crude human nasopharyngeal carcinoma (KB) cell preparations is demonstrated based on characteristic gel filtration elution profiles of M-FBP and S-FBP (Ve/V0 = 1.3 and 1.7, respectively) in Triton X-100. M-FBP is stoichiometrically converted to S-FBP in a time- and temperature-dependent reaction by a metalloprotease which is: heat-labile; particulate; contained in human KB cell and placental membranes, and rat kidney homogenates; inhibited by EDTA, 1,10-phenanthroline, and parahydroxymercuribenzoate; requires divalent cations; is maximally active at neutral pH; and is active in the presence or absence of detergent. The purified soluble FBP product appears to be identical to S-FBP. Conversion of purified endogenously [3H]leucine-labeled M-FBP yields a soluble FBP characterized by a 45% decrease in specific activity (moles of 3H/mol folate bound) relative to M-FBP and a non-folate binding fragment which contains 45% of the [3H]leucine from M-FBP, requires detergent and/or urea to remain soluble, and migrates aberrantly on gel filtration in 1% (v/v) Triton X-100 and 8 M urea. Based on changes in the specific activity and the gel filtration elution profiles of purified labeled M-FBP associated with conversion to S-FBP, the endoproteolytic cleavage site is predicted between residues 226 and 229 of the cDNA predicted human FBP amino acid sequence. These results suggest that the cDNA predicted hydrophobic carboxyl terminus (residues 227-257) remains intact on the fully processed, membrane-anchored M-FBP, contains the Triton binding domain, and is involved in the formation of the membrane anchor of M-FBP.

Folate and methotrexate interactions in the rat kidney.

The mechanisms controlling the renal retention and urinary output of methotrexate and folates were studied in rats. 125I-Labeled folic acid administered i.v. was shown to be retained in the kidney through a system that could be inhibited by either folic acid or 5-methyltetrahydrofolate. Methotrexate also inhibited this system but required concentrations 50- to 100-fold greater than that required for folic acid and 5-methyltetrahydrofolate. Extracts from solubilized kidneys were shown to contain a folate binder with the same relative affinities for folates and methotrexate as the in vivo system. Methotrexate was shown to cause an increase in the urinary output of endogenous folates in rats when administered as equivalent doses to those used in treating human disease. Conversely, [3H]methotrexate administered i.v. was shown to be retained in the kidney through an additional system that could be inhibited by unlabeled methotrexate, but not by folates. This system was not demonstrable in solubilized kidney. These data demonstrate two systems for the renal retention of methotrexate.