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PubMed2003

Yeast Fms1 is a FAD-utilizing polyamine oxidase.

In this report we show that recombinant Saccharomyces cerevisiae Fms1 protein is a polyamine oxidase that binds FAD with an FAD:Fms1 stoichiometry of 1:1. Biochemical characterization of Fms1 shows that it can oxidize spermine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine, but not spermidine. The products of spermine oxidation are spermidine and 3-aminopropanal. A kinetic analysis revealed that spermine, N(1)-acetylspermine, and N(1)-acetylspermidine are oxidized with similar efficiencies, while N(8)-acetylspermidine is a poor substrate. The data support a previous report, suggesting that Fms1 is responsible for the production of beta-alanine from spermine for the synthesis of pantothenic acid.

Acetylation↗
PubMed2000

Pantetheinase activity of membrane-bound Vanin-1: lack of free cysteamine in tissues of Vanin-1 deficient mice.

Pantetheinase (EC 3.5.1.-) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti-oxidant. We show that the Vanin-1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin-1 transfectants and is immunodepleted by specific antibodies; (2) Vanin-1 is a GPI-anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin-1 null mice are deficient in membrane-bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane-bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin-1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.

Amidohydrolases↗
PubMed1987

Mitochondrial synthesis of coenzyme A is on the external surface.

Synthesis of coenzyme A (CoA) in isolated rat heart mitochondria was studied. Mitochondria synthesized CoA from 4'phosphopantetheine (4'PP), a precursor of CoA which is synthesized from pantothenic acid in the cytosol. The synthesis was time dependent and was absolutely dependent on the presence of external ATP under the conditions used. The rate of synthesis was increased either by increasing the pH from 7.4 to 8.5 or by adding 0.01% deoxycholate to the incubation medium. To determine whether the synthesis was intra- or extramitochondrial, mitochondria were separated from the incubation mixture by centrifugation and assayed for CoA. The amount of CoA appearing in the supernatant after 30 mins of incubation increased with increasing concentrations of 4'PP while that in the mitochondrial pellet remained constant over the concentration range studied. Synthesis of CoA from 4'PP was not affected by the uncoupler 2,4-dinitrophenol, or by carboxyatractyloside, or by a combination of the two drugs. The combination was used in an effort to deplete intramitochondrial ATP and to prevent external ATP from entering the mitochondria, thus resulting in mitochondria devoid of matrix ATP. The absolute dependence of synthesis on external ATP, the appearance of newly synthesized CoA in the incubation buffer and the ability of mitochondria lacking matrix-ATP to synthesize CoA suggest that the mitochondrial enzymes responsible for synthesis of CoA from 4'PP are on the outer membrane or on the outer side of the inner membrane.

Adenosine Triphosphate↗
PubMed2000

Content of minor and trace elements, and organic nutrients in representative mixed total diet composites from the USA.

As part of an international nutrition project sponsored by the International Atomic Energy Agency (see footnote), Vienna, Austria, a number of bench-mark mixed total diet composites from the United States were collected and analyzed for minor and trace elements. In this segment of the project, the daily dietary intakes of the minor elements Ca, Cl, K, Mg, N and P and the trace elements Al, As, Au, Br, Cd, Co, Cr, Cs, Cu, Fe, Hg, I, Mn, Mo, Ni, Pb, Rb, Sb, Sc, Se, Sn, Sr, V, Zn and W were determined in mixed total diet composites of foods collected in the FDA Total Diet Study (FDA-TDS). These diets are representative of foods consumed by 25-30-year-old males (representing the mixed population groups in the United States), the highest of eight intake groups in the TDS scheme. In order to link the US mixed diet composite results from this study group to the more comprehensive information generated by the FDA-TDS, the results are compared with the same age-sex group published by the FDA-TDS scheme. The FDA-TDS scheme is based on individual analysis of the 201 food items, with resultant calculation of the daily intake representative of various age-sex groups. The comparison shows excellent agreement for 21 elements which have been investigated by both approaches. Additional elements are reported in the US mixed diet composites from the present study which demonstrate a valuable supplement to the data obtained by FDA-TDS scheme. Further the mixed total diet composite approach has also proven useful for the assessment of dietary intake of proximates (protein, fat, carbohydrates), fiber and phytate. In addition, vitamins thiamin, riboflavin, niacin, B6, B12, pantothenic acid, folic acid and biotin were also assayed in these composites.

Adult↗
PubMedDate not supplied

Dietary intake of community-based HIV-1 seropositive and seronegative injecting drug users.

Dietary intake was assessed in a subsample of a cohort of inner-city injecting drug users (IDUs). In this population of predominantly African-American IDUs, including both HIV -1-infected and noninfected men and women, a food frequency questionnaire (FFQ) and a 24-h recall were administered. One hundred seven volunteers participated. Although total caloric intake was consistently higher with the food frequency method, percent of total calories from fat, protein, and carbohydrates were similar between the FFQ and 24-h recall. Spearman's correlations for agreement between the 24-h recall and the FFQ ranged from 0.22 for vitamin E to 0.52 for carbohydrates. HIV-1 seropositives reported higher protein (p = 0.05) and fat (p = 0.02) consumption than seronegatives according to the 24-h recall. The difference in total fat consumption was due to higher intakes of saturated and monounsaturated fats (p = 0.01). Median intakes of vitamins B2 and B12, pantothenic acid (p < or = 0.05), phosphorous (p < or = 0.01), and selenium (p < or = 0.005) were also greater in HIV-1 seropositives. Reported intake of vitamins A and E, calcium, and zinc were below the recommended daily allowances for both HIV-1 seropositives and seronegatives. Although intakes of most nutrients appeared adequate for the group as a whole, extreme ranges were observed and may be the result of imbalanced food selections and day to day variation. Food group analysis indicated low intakes of fruits, vegetables, milk, and milk products. More research is needed to fully understand the implication of dietary habits and nutritional status in the free-living HIV-1-infected and noninfected IDUs.

Adult↗
PubMed2000

Oral vitamin intake in children receiving long-term dialysis.

OBJECTIVE: To evaluate dietary and oral supplement vitamin intake in children submitted to dialysis (peritoneal dialysis and hemodialysis). DESIGN: Prospective clinical trial in a 12-month follow-up period. SETTING: Children with end-stage renal disease (ESRD) who attended the pediatric nephrology clinic of Universidade Federal de São Paulo-Escola Paulista de Medicina (UNIFESP-EPM), São Paulo, Brazil. PATIENTS: Thirty children (18 girls, 23 in peritoneal dialysis, 7 in hemodialysis) with age 9.3 +/- 7.4 years. INTERVENTION METHODOLOGY: Six successive assessments of both anthropometric indexes and 3-day dietary diaries in children receiving a daily dose of oral water-soluble vitamin supplement. MAIN OUTCOME MEASURES: Anthropometric indexes (weight/age [W/A], height/age [H/A], midarm muscle area/age [MAMA/A], and fat area/age [FA/A]) and dietary adequacy-% recommended dietary allowance (RDA) (computerized nutritional analysis from 3-day dietary intake diary). RESULTS: Anthropometric indexes analysis showed that 53% of children were <-2.0 standard deviation score (SDS) of W/A, 63% were <-2.0 SDS of H/A, and 43.3% were <-1.65 SDS of MAMA/A, suggesting growth deficit and low muscle wasted. Total caloric intake was lower than 100% of RDA in 90% of children. Dietary intake of water-soluble vitamins was <100% of RDA in the majority of children, as follows: vitamin C (24/30), B1 (28/30), B2 (22/30), B3 (27/30), B6 (26/30), B12 (1/30), pantothenic acid (24/30), and folic acid (9/30). The combined dietary and vitamin supplement intake resulted in excessive oral intake for almost all the vitamins. CONCLUSION: Dietary intake of water-soluble vitamins is lower than the RDA in the majority of children with ESRD; supplementation is necessary to reach the RDA. The use of the available vitamin supplement resulted in vitamin intakes that exceeded the RDA for almost all of the vitamins. However, we do not know if these intakes exceeded the children's requirements, nor whether they had any clinically significant harmful effects.

Adolescent↗
PubMedDate not supplied

Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli.

Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly ( approximately 10-fold). The acetyl-CoA levels also increased ( approximately 5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.

Acetyl Coenzyme A↗
PubMedDate not supplied

Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate.

Although the bacterium E. coli is chosen as the host in many bioprocesses, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that could have otherwise been used for the desired product. The production of the ester isoamyl acetate from acetyl-CoA by ATF2, a yeast alcohol acetyl transferase, was used as a model system to demonstrate the beneficial effects of reducing acetate production. All strains tested for ester production also overexpressed panK, a native E. coli gene that previous studies have shown to increase free intracellular CoA levels when fed with pantothenic acid. A recombinant E. coli strain with a deletion in ackA-pta produces less acetate and more isoamyl acetate than the wild-type E. coli strain. When both acetate-producing pathways were deleted, the acetate production was greatly reduced. However, pyruvate began to accumulate, so that the overall ester production remained largely unchanged. To produce more ester, a previously established strategy of increasing the flux from pyruvate to acetyl-CoA was adopted by overexpressing pyruvate dehydrogenase. The ester production was then 80% higher in the poxB, ackA-pta strain (0.18 mM) than that found in the single ackA-pta mutant (0.10 mM), which also overexpressed PDH.

Chromatography, High Pressure Liquid↗
PubMed2006

Simultaneous determination of eight water-soluble vitamins in supplemented foods by liquid chromatography.

A fast, simple, and reliable method for the isolation and determination of the vitamins thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, folic acid, cyanocobalamin, and ascorbic acid in food samples is proposed. The most relevant advantages of the proposed method are the simultaneous determination of the eight more common vitamins in enriched food products and a reduction of the time required for quantitative extraction, because the method consists merely of the addition of a precipitation solution and centrifugation of the sample. Furthermore, this method saves a substantial amount of reagents as compared with official methods, and minimal sample manipulation is achieved due to the few steps required. The chromatographic separation is carried out on a reverse phase C18 column, and the vitamins are detected at different wavelengths by either fluorescence or UV-visible detection. The proposed method was applied to the determination of water-soluble vitamins in supplemented milk, infant nutrition products, and milk powder certified reference material (CRM 421, BCR) with recoveries ranging from 90 to 100%.

Animals↗
PubMed2003

Malnutrition in institutionalized seniors: the iatrogenic component.

OBJECTIVES: To provide practitioners with benchmarks for micronutrient supplementation policies for older residents of long-term care (LTC) facilities, based upon residents' usual energy intake. DESIGN: Retrospective secondary analysis of nutrient intake and delivery data, obtained from 21 consecutive collection days. SETTING: A fully accredited geriatric teaching facility, affiliated with the University of Toronto's Medical School, that includes a chronic care hospital and a home for the aged, housing more than 800 senior residents. PARTICIPANTS: Twenty-three cognitively impaired residents who retained the ability to feed themselves. MEASUREMENTS: Average daily micronutrient (vitamin and mineral) content of two common diet types--unrestricted diet as tolerated and lactose-free--were evaluated based upon all foods served during a 28-day cycle of menus, providing an estimate of total possible micronutrient intake. Energy intake needed to meet recommended intakes for each of the micronutrients was then determined and compared with actual dietary intakes. RESULTS: Even if entirely consumed, neither diet (providing approximately 2,000 kcal/d) supplied sufficient quantities of vitamins (vitamin E, pantothenic acid) and minerals (calcium, zinc, copper and manganese) to enable residents to meet recommended intakes, making these deficiencies iatrogenic in nature. As projected intake levels fell to reflect ranges more consistently observed in LTC residents (1,000-1,500 kcal/d), the number and severity of risk nutrients increased. Similar predictions, based upon actual intake measurements, were made and confirmed. CONCLUSIONS: The traditional approach of developing LTC menus using resources such as Canada's Food Guide or the Food Guide Pyramid of the United States results in iatrogenic malnutrition. New guidelines are needed that are targeted toward the special needs of older adults who have low food intakes. In the interim, all older LTC residents require full-spectrum vitamin and mineral supplements.

Aged↗
PubMed2005

Micronutrient dilution associated with added sugar intake in elderly black South African women.

OBJECTIVES: To investigate the association between added sugar and macronutrient and micronutrient intakes, and to assess whether added sugar intake is related to biochemical indices of nutritional status, Mini-Nutritional Assessment (MNA) score, body mass index (BMI) and performance on physical function tests. DESIGN: A cross-sectional, analytical study. SETTING AND SUBJECTS: Convenient sample of 285 institutionalised and community-dwelling black South African men and women aged 60+ y. METHODS: An interviewer-administered 24-h dietary recall and MNA were performed. Serum albumin, vitamin B12, ferritin, cholesterol, haemoglobin, red blood cell (RBC) folate and plasma vitamin C were measured. Handgrip strength, BMI, 'sit-to-stand' and 'get-up-and-go' tests were measured. Outcome variables were analysed according to tertiles of added sugar, in grams and as a percentage of total energy (% E). RESULTS: In each tertile of sugar intake, mean MNA score fell in the 'at-risk' classification. In women, energy, protein, % E protein, fibre, thiamin, riboflavin, niacin, vitamin B6, folate, pantothenic acid, biotin, vitamin C, calcium, iron, magnesium, phosphorus, zinc, copper and selenium intake were significantly lowest in subjects in the highest % E sugar tertile. In men, no differences were found for micronutrient intake according to tertiles of total added sugar or % E added sugar. Physical function declined with increasing sugar intake, and suboptimal RBC folate and plasma ascorbic acid status was associated with increasing sugar intake (in women). No relationship was found between added sugar intake and the very high prevalence (65%) of obesity in women. CONCLUSION: A nutrient-diluting effect of added sugars intake was demonstrated in elderly black South African women. Further studies in this population are required in order to develop food-based dietary guidelines, which include messages on added sugar intake.

Activities of Daily Living↗
PubMed2002

Newly established regulation in Japan: foods with health claims.

The Japanese government enacted a new regulatory system called 'Foods with health claims' in April 2001, which consists of 'Foods for Specified Health Use' (FOSHU) and 'Foods with Nutrient Function Claims' (FNFC). The FOSHU was set up by the Ministry of Health and Welfare in 1991 to approve descriptions on a label regarding an effect of food on the human body. It was enacted as a part of 'food for specified dietary use' under the Nutrition Improvement Law. There are three important requirements for FOSHU approval. The first is scientific evidence of the efficacy, including clinical testing. The second is safety for consumption. The third is analytical determination of the effective component. At present there are 293 items approved as FOSHU. Most of the descriptions of foods under the FOSHU system are similar to the category of enhanced function claims of Codex. Under FNFC, 12 vitamins (vitamin A, B1, B2, B6, B12, C, E, D, biotin, pantothenic acid, folic acid, and niacin) and two minerals (calcium and iron) are standardized. These claims are similar to the nutrient function claims approved by Codex in 1997. It is desirable that the Japanese administration and the food industry cooperate with ASEAN countries to work together in the development and promotion of nutrition and health claims on foods.

Food↗
PubMed1999

Four intracellular genomes direct weevil biology: nuclear, mitochondrial, principal endosymbiont, and Wolbachia.

Cell physiology in the weevil Sitophilus oryzae is coordinated by three integrated genomes: nuclear, mitochondrial, and the "S. oryzae principal endosymbiont" (SOPE). SOPE, a cytoplasmic bacterium (2 x 10(3) bacteria per specialized bacteriocyte cell and 3 x 10(6) bacteria per weevil) that belongs to the proteobacteria gamma3-subgroup, is present in all weevils studied. We discovered a fourth prokaryotic genome in somatic and germ tissues of 57% of weevil strains of three species, S. oryzae, Sitophilus zeamais, and Sitophilus granarius, distributed worldwide. We assigned this Gram-negative prokaryote to the Wolbachia group (alpha-proteobacteria), on the basis of 16S rDNA sequence and fluorescence in situ DNA-RNA hybridization (FISH). Both bacteria, SOPE and Wolbachia, were selectively eliminated by combined heat and antibiotic treatments. Study of bacteria involvement in this insect's genetics and physiology revealed that SOPE, which induces the specific differentiation of the bacteriocytes, increases mitochondrial oxidative phosphorylation through the supply of pantothenic acid and riboflavin. Elimination of this gamma3-proteobacterium impairs many physiological traits. By contrast, neither the presence nor the absence of Wolbachia significantly affects the weevil's physiology. Wolbachia, disseminated throughout the body cells, is in particularly high density in the germ cells, where it causes nucleocytoplasmic incompatibility. The coexistence of two distinct types of intracellular proteobacteria at different levels of symbiont integration in insects illustrates the genetic complexity of animal tissue. Furthermore, evolutionary timing can be inferred: first nucleocytoplasm, then mitochondria, then SOPE, and finally Wolbachia. Symbiogenesis, the genetic integration of long-term associated members of different species, in the weevil appears to be a mechanism of speciation (with Wolbachia) and provides a means for animals to acquire new genes that permit better adaptation to the environment (with SOPE).

Biological Evolution↗
PubMed2000

Structural basis for the feedback regulation of Escherichia coli pantothenate kinase by coenzyme A.

Pantothenate kinase (PanK) is a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway and catalyzes the phosphorylation of pantothenic acid to form phosphopantothenate. CoA is a feedback inhibitor of PanK activity by competitive binding to the ATP site. The structures of the Escherichia coli enzyme, in complex with a nonhydrolyzable analogue of ATP, 5'-adenylimido-diphosphate (AMPPNP), or with CoA, were determined at 2.6 and 2.5 A, respectively. Both structures show that two dimers occupy an asymmetric unit; each subunit has a alpha/beta mononucleotide-binding fold with an extensive antiparallel coiled coil formed by two long helices along the dimerization interface. The two ligands, AMPPNP and CoA, associate with PanK in very different ways, but their phosphate binding sites overlap, explaining the kinetic competition between CoA and ATP. Residues Asp(127), His(177), and Arg(243) are proposed to be involved in catalysis, based on modeling of the pentacoordinate transition state. The more potent inhibition by CoA, compared with the CoA thioesters, is explained by a tight interaction of the CoA thiol group with the side chains of aromatic residues, which is predicted to discriminate against the CoA thioesters. The PanK structure provides the framework for a more detailed understanding of the mechanism of catalysis and feedback regulation of PanK.

Adenosine Triphosphate↗
PubMed2002

Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics.

The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.

Blotting, Northern↗
PubMed2003

Risk analysis applied to food fortification.

OBJECTIVE: To describe how a risk analysis can be applied to food fortification, with emphasis on voluntary fortification and intake levels that might exceed usual dietary levels. DESIGN: Use of the risk analysis model as a frame to classify nutrients according to the risk of exceeding upper safe intake levels. Furthermore, to apply the model when discussing possible consequences of liberal fortification practices on eating behaviour and disease patterns. SETTING: The discussion on food fortification presently going on internationally. RESULTS: Micronutrients can be classified according to their safety margin, i.e. the size of the interval between the recommended intake and the upper safe level of intake. We suggest that nutrients with a small safety margin, i.e. for which the upper safe level is less than five times the recommended intake, be placed in a category A and should be handled with care (retinol, vitamin D, niacin, folate and all minerals). Category B comprises nutrients with an intermediate safety margin (vitamins E, B6, B12 and C), while nutrients that according to present knowledge are harmless even at 100 times the recommendation (vitamin K, thiamin, riboflavin, pantothenic acid and biotin) are categorised as C. DISCUSSION: The risk analysis model is a useful tool when assessing the risk of both too low and excess intakes of single micronutrients, but can also be applied to analyse the consequences of fortification practices on eating behaviour and disease patterns. Liberal fortification regulations may, for example, distort the conception of what is healthy food, and drive consumption towards a more unhealthy diet, contributing to the plague of overweight and concomitant increased risk of degenerative diseases. CONCLUSION: The impact of fortification practices on the total eating pattern of a population should become an integrated part of the discussions and regulations connected to the issue.

Food, Fortified↗