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Biomedical subjects

D B McCormick

Publications and source records attributed to D B McCormick.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of a new flavin metabolite from human urine.

A new flavin metabolite comprising approximately 5% of the total flavin of human urine was isolated and characterized using absorption and fluorescence spectra, oxidation-reduction and hydrolysis data, and ninhydrin reactions. The flavin is a derivative associated with a peptide residue in ester linkage from an amino acid carboxyl to the ribityl chain of riboflavin, probably at the 5'-terminus.

Adult↗
PubMed1987

Complete purification and general characterization of FAD synthetase from rat liver.

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.

Animals↗
PubMed1987

Flavin catabolites: identification and quantitation in human urine.

Riboflavin is the primary flavin excreted in human urine but significant amounts of 7 alpha-hydroxyriboflavin and lesser amounts of 8 alpha-hydroxyriboflavin are present and reflect tissue microsomal oxidations. A newly found flavin catabolite of an 8 alpha-sulfonyl type may reflect intake and/or turnover of such thioether-linked flavin as occurs in monoamine oxidase. Additionally, lesser amounts of 10-hydroxyethylflavin (indicative of intestinal microbial action on the vitamin) and traces of lumiflavin (arising from photodecomposition) constitute part of the remaining flavin, which acutely reflects level of intake.

Adult↗
PubMed1987

Clarification and quantitation of primary (tissue) and secondary (microbial) catabolites of riboflavin that are excreted in mammalian (rat) urine.

Riboflavin derivatives were quantitated and identified in urine of rats fed 0, 2 and 6 micrograms riboflavin/g diet per day both with and without added succinyl sulfathiazole for 6 wk. Two rats from each dietary group were placed in metabolic cages and urine was collected in the dark for 24 h. On the fourth week, a third animal from each group received an i.p. injection of [2-14C]riboflavin before being placed in a metabolic cage and urine collected in the dark for 48 h. Urine samples were extracted with phenol for flavin components and with chloroform for lumichrome and derivatives. Riboflavin was the predominant flavin excreted by rats in all dietary groups, followed by hydroxymethylriboflavins and smaller amounts of flavin mononucleotide (FMN), lumiflavin and 10-hydroxyethylflavin. Carboxylumichromes accounted for 5-10% of the total flavin-derived fluorescence in urine of rats fed 2 and 6 micrograms riboflavin/g diet and were reduced to approximately 3% when sulfathiazole was added to the base diets. Carboxylumichromes were absent from urine of riboflavin-deficient rats. Riboflavin accounted for 85-90% of the recovered radioactivity of all radioactive urine extracts; no radioactively labeled carboxylumichromes were detected. These results indicate that hydroxymethylriboflavins are primary catabolites of riboflavin derived from tissue microsomal oxidations, whereas carboxylumichromes reflect the continued oxidation of ring hydroxymethyl functions plus gut microbial cleavage of the side chain of flavin.

Animals↗
PubMed1986

Immunoglobulins associated with elevated riboflavin binding by plasma from cancer patients.

Plasma from 182 patients with different malignant diseases was tested for riboflavin binding by immunoglobulins, which have been recently identified as major carriers of this micronutrient. A wide range of binding (5.9 to 130 pmole/ml plasma) was observed, and significant elevations were found for patients having breast cancer (21.2 +/- 1.9, P less than 0.05) and melanoma (25.7 +/- 1.9, P less than 0.001) compared to controls (15.5 +/- 1.9). The proteins responsible for a majority of the higher binding were identified as immunoglobulins, based on their elution from gel filtration columns and the removal of 57-88% of the non-albumin binding by treating of plasma with Protein A-agarose. The binding was only weakly related to the total concentration of immunoglobulins (r = 0.11 by linear regression analysis), however, and is apparently due to a subclass that is elevated in some types of cancer. Elevated levels of these immunoglobulins may contribute to the lower urinary levels and clearance of riboflavin in cancer.

Breast Neoplasms↗
PubMed1986

Marital mores as a mechanism for the maintenance of ethnic variations of lethal gene frequencies.

A computer simulation model was developed to study the effects of various marital mores on the incidence of lethal autosomal recessive genes in populations that are subdivided into small isolates. The problem was studied in isolates where initial generation size was 30, 40, and 50 individuals. In each of these, the mean fertility rate was varied from 2.3 to 2.7 surviving (to adulthood) children per couple whose marriage had been contracted in accordance with the prevailing convention: marriage between first cousins and siblings prohibited; marriage between siblings prohibited; marriage allowed between any individuals; marriage prohibited between siblings but encouraged between cousins; and marriage encouraged between siblings. In all cases, the mean gene frequency in generation 20 was lower than that predicted by the deterministic model with random mating in an unsubdivided population of infinite size, due to gene loss through random drift (to zero) in many of the isolates. The mores that encouraged consanguineous marriages had the lowest final lethal-gene frequencies. Random mating produced intermediate values, and the restrictive mores, the highest final frequencies. The deterministic model (assuming infinite population size and random mating) predictions of the final gene frequency were exceeded only if there was reproductive compensation. It is concluded that restrictive marital mores significantly reduce the selective pressures on lethal recessive genes in small isolates, but that this is counteracted by the increased rate of gene loss through random drift.

Computer Simulation↗
PubMed1985

Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate.

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.

Animals↗
PubMed1985

High-performance liquid chromatography of biotin and analogues.

Biotin, analogues, and chemical intermediates were separated by high-performance liquid chromatography (HPLC) using reversed-phase and anion-exchange chromatographic conditions. Reversed-phase separations provided a wide range of retention times and resolution of nearly all the biotin compounds from mixtures of the analogues. Anion-exchange separations gave generally shorter retention times as compared to reversed-phase separations and greater resolution between biotin l- and d-sulfoxide. However, fewer analogues were resolved from mixtures of the compounds with anion-exchange HPLC.

Biotin↗
PubMed1985

Thyroid hormone regulation of flavocoenzyme biosynthesis.

The means by which thyroid hormone regulates flavocoenzyme biosynthesis was studied in hyper-, eu-, and hypothyroid rats by determining the activities of flavocoenzyme-forming enzymes, viz., flavokinase and FAD synthetase, as well as those of flavocoenzyme-degrading enzymes, viz., FMN phosphatase and FAD pyrophosphatase. Flavokinase activity was increased in hyperthyroid animal and decreased in hypothyroid animals. Correspondence of flavokinase activity with the amount of a high-affinity flavin-binding protein quantitated immunologically in hypo-, eu-, and hyperthyroid rats indicated that the thyroid response is caused by an increased amount of enzyme; moreover, the concomitant decrease in a low-affinity flavin-binding protein suggests an inactive precursor form of flavokinase. FAD synthetase activity showed a similar but less pronounced trend than flavokinase. Activities of FMN phosphatase and FAD pyrophosphatase were not influenced by thyroid hormone. Overall results indicate that the mechanism of thyroid hormone regulation of flavocoenzyme level is in the steps of biosynthesis, especially at flavokinase, rather than in degradation steps.

Animals↗
PubMed1985

Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible.

Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient.

Autoradiography↗
PubMed1985

Urinary lumichrome-level catabolites of riboflavin are due to microbial and photochemical events and not rat tissue enzymatic cleavage of the ribityl chain.

The occurrence of 7- and 8-carboxylumichromes as a significant fraction of riboflavin-derived material in rat urine has led to a reexamination of tissues for possible enzymatic activity that could contribute to cleavage at position 10 of the D-ribityl chain of the vitamin. For this, homogenates were incubated in the dark under various conditions with pure [2-14C]riboflavin with and without cofactors prior to extractions with phenol for flavins or chloroform for lumichromes, and high pressure liquid chromatography was used to separate and to quantitate all radioactive compounds. Only unmodified riboflavin and small amounts of flavin mononucleotide (FMN) were found even though the level of detection of known derivatives including 10-(2'-hydroxyethyl)flavin and lumichrome was shown to be extremely sensitive. Hence, rat tissue per se does not appear responsible for significant cleavage of the D-ribityl chain of riboflavin. Rather such cleavage to the lumichrome level must result from the known actions of intestinal microflora and potentially from photolysis.

Animals↗
PubMed1984

Mechanism of pyridoxine uptake by isolated rat liver cells.

Uptake of [3H]pyridoxine by isolated rat hepatocytes was detected at substrate concentrations as low as 0.5 microM. At this concentration, an initial uptake rate was 1.87 +/- 0.17 pmol/10(6) cells X min at 37 degrees C. Both the initial and a subsequent much slower pyridoxine accumulation were strongly inhibited by low temperature as well as by 10 mM ethionine, which competes for available ATP, or 1 microM carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which inhibits oxidative phosphorylation and the supply of ATP. The uptake process is apparently insensitive to 1 mM ouabain and is Na+ independent. The initial uptake rate was saturable at higher concentrations of [3H]pyridoxine with an apparent Km of 28 +/- 8 microM and Vmax of 106 +/- 27 pmol/10(6) cells X min. The Km value corresponds to that reported for pyridoxine as a substrate of pyridoxal kinase. Moreover, the transport process was inhibited by structural analogs of [3H]pyridoxine even at concentrations equimolar to the normal substrate. The established order of inhibitory effectiveness, 4'-deoxypyridoxine greater than unlabeled pyridoxine greater than 5'-deoxypyridoxine, is in agreement with known properties of the kinase. It is concluded that pyridoxine uptake probably occurs by diffusion, simple or facilitated, followed by metabolic trapping due to pyridoxal kinase-catalyzed phosphorylation. Pyridoxine deficiency had no significant effect on uptake of this B6 vitamer by hepatocytes.

Adenosine Triphosphate↗