The hydrolysis of these high - energy phosphate bonds release energy which powers cellular energy requiring processes medicine 831 purchase phenytoin 100mg without prescription. Energy of hydrolysis of thioester bond is mostly used to drive the reactions forward to completion medicine used for adhd phenytoin 100 mg discount. For example treatment bursitis order phenytoin 100mg fast delivery, the energy of chemical bonds of carbohydrates treatment goals for depression buy cheap phenytoin 100mg line, lipids and proteins is released and captured in utilization form by processes involving oxidation- reductions. In biological systems the primary electron donors are fuel molecules such as carbohydrates, fats and proteins. This occurs by the help of energy conserving system in the inner mitochondrial membrane of eukaryotes or plasma membrane of prokaryotes. This metabolic pathway in addition to providing energy provides building blocks required for growth, reproduction, repair and maintenance of cellular viability. Structurally it is bounded by two separate membranes (outer mitochondrial membrane and inner mitochondrial membrane) Out membrane - smooth and unfolded - Freely permeable to most ions and polar molecules (Contain porous channels) Inner membrane - folded into cristae-increased surface area - Highly impermeable to most ions and polar molecules Contain transporters which access polar and ionic molecules in and out Cristae are characteristic of muscle and other metabolically active cell types - Protein-rich membrane (about 75%) Inter membrane space space between outer and inner membranes Matrix-the internal compartment containing soluble enzymes and mitochondrial genetic material Fig 3. Inside matrix pyruvate is oxidized into acetylCoA by pyruvate dehydrogenase complex which is complex of E1, E2 and E3 enzymes. Reactions take place in cytosol of prokaryotes and mitochondria matrix of eukaryotes 63 Fig 3. Considerable free energy is lost as heat due to hydrolysis of thisester bond (drive the reaction forward). This enzyme has the same coenzyme requirement to that of pyruvate dehydrogenase complex. Transfer and accept two electrons at a time Cytochromes heme conjugated proteins Heme = Fe2+/F3+ + porphyrin Include classes of cytochromes designated a, b, and c. Iron at the center of cytochromes accept and donates single electron Cytochrone Fe2+ (reduced) Cytochrome Fe3+ + e(Oxidized) Cytochrome with relatively less positive reduction potential. CoQ and cytochrome C are mobile electron carriers which act as a link between the complexes. The free energy released is captured at three sites to pump protons against concentration gradient from matrix to inter membrane space generating proton gradient across inner mitochondrial membrane. As a result of this pH gradient is also formed, more positive (acidic) on the outer side more negative (basic) on the inner side of mitochondria. Now the kinetic energy of electrons is transformed into the proton motive force. They also dissolve the vitamins, which are fat-soluble and assist their digestion. Complex lipids:- Esters of fatty acids and alcohols together with some other head groups. Glycerophospholipids:- the alcohol is glycerol Sphingophospholipids:- the alcohol is shingosine. Glycolipids:- Lipids containing fatty acid, sphingosine and carbohydrate residues. Others:- Include sulfolipids, amino lipids and lipoproteins, which are modified forms of lipids. The simplest naturally occurring lipids are triacylglycerols formed by esterification of fatty acids with glycerol. Mostly the double bond occurs at the 9th carbon as we count from the carboxyl group end. In a different way the position of the double bond(s) can be indicated as shown in the second expression without the delta. C18:1, 9 or 18:1(9) C18 indicates 18 carbons, 1 indicates the number of double bonds, delta 9(9) indicates the position of double bond between 9th and 10th carbon atoms. Double bonds in naturally occuring fatty acids are in the cis- configuration and saturated fatty acids of C12 to C24 are solids at body temperature but the unsaturated once are liquids. Poly unsaturated fatty acids are released from membranes, diverted for the synthesis of prostaglandins, leukotriens and thromboxanes. They act as fat mobilizing agents in liver and protect liver from accumulating fats (fatty liver). Triacylglycerols or also called as triacylglycerides, exist as simple or mixed types depending on the type of fatty acids that form esters with the glycerol. Both saturated and/or unsaturated fatty acids can form the ester linkage with the backbone alcohol. Triacylglycerols are mainly found in special cells called adipocytes (fat cells), of the mamary gland, abdomen and under skin of animals. Structure of phosphatidate Phosphatidate is the parent compound for the formation of the different glycerophospholipids. If choline is attached it is called phosphatidyl choline (lecithin), if ethanolamine is attached it is called phosphatidyl ethanolamine. The second largest membrane lipids are sphingolipids, which contain two non-polar and one polar head groups. One unit shall definitely be N-acetyl neuraminic acid (sialic acid) 6% of grey brain matter is ganglioside. Cerebrosides:- these are glycolipids which have no phosphate group but neutral head group and contain one or two sugar groups usually glucose or Galactose Functions of phospholipids 1. Phospholipids are components of membrane; impart fluidity and pliability to the membrane. Dipalmitoyl choline (lecithin) acts as surfactant and lowers the surface tension in alveoli of lungs. Lecithin along with sphingomyelin maintains the shape of alveoli and prevents their collapse due to high surface tension of the surrounding medium. Steroids are complex fat-soluble molecules, which are present in the plasma lipoproteins and outer cell membrane. Cholesterol is one of the important non fatty acid lipid that is grouped with steroids. Cholesterol is important in many ways: · For the synthesis of bile salts that are important in lipid digestion and absorption. Digestion and Absorption of Lipids Diet contains triglycerides, cholesterol and its ester, phospholipids, fattyacids etc. Thus 3 fatty acids and one glycerol molecule is produced from the digestion of dietary triglyceride. Phospholipase B acts on Lysophospholipid, produces glycerophosphoryl choline and free fatty acid. Cholesterol esterase hydrolyses cholesterol ester to free cholesterol and one fatty acid. Cholesterol, long chain fatty acids are esterified and absorbed in form of micelles. Impaired secretion of lipases from the pancreas and bile salts from liver results in failure in fat absorption and causes steatorrea (excessive passage fatty stool). The micelles, through the intestinal lumen move to the brush border of the mucosal cells where they are absorbed into the intestinal epithelium. The free fatty acids and monoayclglycerols are absorbed through the epithelial cells lining the small intestine and pass to the lymphatic system where they join the systemic blood via the thoracic duct. The free fatty acids in blood (long chain) are bound to albumin and transported by blood to the liver. Metabolism of Fatty Acids and Triacylglycerols the triacylglycerols play an important role in furnishing energy in animals. They provide more than half the energy need of some organs like the brain, liver, heart and resting skeletal muscle. Mobilization of Fatty Acids from Adipocytes When the energy supply from diet is limited, the body responds to this deficiency through hormonal signals transmitted to the adipose tissue by release of glucagon, epinephrine, or adrenocorticotropic hormone. The glycerol produced is taken up by liver, phosphorylated and oxidized to dihydroxyacetone phosphate, which is isomerised to glyceraldehydes-3-phosphate, an intermediate of both glycolysis and gluconeogenesis. Therefore, the glycerol is either converted to glucose (gluconeogenesis) or to pyruvate (glycolysis). The reaction is catalyzed by AcylCoA synthetase or also called thiokinase, found in the cytosol and mitochondria of cells. The transport of acyl derivatives across the mitochondrial membrane needs three acyltransferases (shuttles). Specific for short chain acyl groups, does not require carnitine Specific for the long chain acyl groups. Therefore, long chain acyl groups cross the mitochondrial membrane in combination with carnitine. Carnitine acyl transferase I, found in the surface of the outer mitochondrial membrane, catalyzes the acyl transferase reaction from acylCoA to the carnitine.
Incubate sample 45 minutes at room temperature Rinse fixed cells 2 times with wash buffer Blocking Add Primary Antibodies Remove blocking buffer symptoms mononucleosis buy phenytoin 100mg, no rinsing is necessary Incubate overnight at 28 °C or for 1 hour at room temperature Rinse 2 times with wash buffer Incubate for 1 hour at room temperature in the dark Add Secondary Antibodies Procedure Note: this protocol is optimized for cells grown on coverslips in a 6- or 24-well plate but can be adapted accordingly medications prescribed for pain are termed 100mg phenytoin amex. Wash the coverslips or slides containing the fixed cells two times in 400 mL of wash buffer medications with pseudoephedrine phenytoin 100 mg for sale. Add 400 mL to the wells and incubate overnight at 28 °C medical treatment 80ddb discount phenytoin 100 mg line, or alternatively, incubate at room temperature for 1 hour. Note: Experimentation with both antibody concentration and incubation time may be required to achieve optimal labeling. Fix the cells with 4% formaldehyde for 20 minutes at room temperature by adding formaldehyde directly to the culture media (see page 5) and adjust to approximately 1 x 106 cells/mL. Pour off the supernatant and resuspend the cell pellet in 200 mL of deionized H2O. Add 5 mL of the cell suspension to a gelatin-coated slide (3 spots per slide) and smear with the side of a pipette tip. Place the slide on a hot plate (low heat setting) and allow the liquid to evaporate. Surround the cell spot with a hydrophobic barrier using a barrier pen and air dry. The slides can be stored at 28 °C for up to 3 months or they may be stained immediately. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (20 to 70 °C) and avoid repeated freeze-thaw cycles. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody. If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody. Repeat staining and confirm that correct reagents are used and are added in the correct order. Problem: High Background Possible Source High concentration of primary and or secondary antibodies. Test or Action Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies. Lower the ionic strength of the antibody diluent (particularly, monoclonal antibodies respond well to reducing the salt concentration). Use an antibody that has had cross-reactive IgG species removed (absorbed against sample species). Problem: Cell/Tissue Morphology Destroyed Possible Source Antigen retrieval methods are too harsh. Underfixation has physically damaged the tissue or cells during the staining process. Test or Action Empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen. Re-cut sections using a sharp blade, or ignore damaged areas when analyzing the results. Problem: Staining is Inappropriate Possible Source Fixation method is inappropriate for the antigen. To learn more about and search for specific R&D Systems brand antibodies, please visit rndsystems. Analysis of Histamine and Tyramine Using Prominence Amino Acid Analysis System We introduce an example of analysis of tyramine and histamine using the Prominence Amino Acid Analysis System, in which detection is conducted using post-column uorescence derivatization. A Comparison Study of Different Capillary Columns for Analysis of Alcohol Congeners in Alcoholic Beverages In this study, four capillary columns are selected and their performance are compared with packed column in terms of separation of all key components found in alcoholic beverages. Comprehensive Two-Dimensional Liquid Chromatograph - Nexera-e Catch the Peaks Behind the Peaks - [Flyer] Developing a Chiral Amino Acid Analysis Method That Uses Column Switching this article introduces a rapid analysis method that employs chiral columns to achieve high separation and high sensitivity and that dispenses with derivatization. Analysis of Chiral Amino Acids within Fermented Beverages Utilizing a Column Switching System this article introduces an example analysis of fermented beverages using a column switching system which alternates between two types of chiral columns using high-pressure column switching valves. However, because they cannot be synthesized in sufficient quantities in the body, if at all, they must be ingested either in our meals or by other means, such as tablets. Vitamins can be classified into two groups, watersoluble and fat-soluble, and their analysis is generally conducted by high-performance liquid chromatography. Of the two types of vitamins, the water-soluble type consists mainly of highly polar basic components which exhibit weak retention in analysis by reversed-phase liquid chromatography. Since they exhibit weak retention, the analysis is typically conducted using ionpair reagents. Using ion-pair reagents can be challenging because of the longer analysis time, widening of slow eluting peaks, difficulty in obtaining sufficient sensitivity and long equilibration times. Analysis efficiency is also compromised due to additional work-flow requirements, such as mobile phase preparation and column conditioning. As folic acid, riboflavin, and biotin are only slightly soluble in water, they were first dissolved in dilute alkali solution, and then dissolved in 10 mmol/L phosphate buffer (sodium) solution (pH 2. T h e re s u l t s u s i n g the S h i m - p a c k M A q C - O D S demonstrate that the analysis time can be shortened due to the use of gradient analysis. This enhances the retention of basic compounds, thereby permitting analysis using a conventional buffer instead of an ionpair reagent such as sodium alkyl sulfonate. It is difficult to improve throughput in ion-pair c h ro m a t o g r a p h y d u e t o the t i m e re q u i re d f o r reequilibration following gradient elution. While on the other hand, in isocratic elution, sensitivity is compromised due to peak broadening from slow eluting substances. As a result, simultaneous analysis of highly polar basic compounds and other components can be achieved while shortening the analysis time and improving sensitivity. These features demonstrate the powerful advantages of this column for simultaneous analysis of watersoluble vitamins, as well as impurities in pharmaceutical products that contain large quantities of basic compounds. Thiamine (Vitamin B1)* Niacin Nicotinamide Pyridoxine (Vitamin B6)* Ca Pantothenate 10 mmol/L Phosphate (sodium) buffer Standard stock solution (500 mg/L each) Folic acid Riboflavin (Vitamin B2) Biotin 20 mmol/L Aqueous sodium hydroxide Standard stock solution (500 mg/L each) 10 mmol/L Phosphate (sodium) buffer Standard solution (20 mg/L each) *Hydrochloride was used for substances indicated with an asterisk. High Performance Liquid Chromatography L467 Analysis of Sugars in Orange Juice and Grape Juice by Prominence-i and Differential Refractive Index Detector Table 1 Analytical Conditions Column Mobile Phase Flowrate Column Temp. Since the column oven can accommodate a 30-cm column for use in sugar analysis (ligand exchange column), and the temperature can be controlled up to 85 °C, it therefore supports applications that require a long column and high column temperature. Calibration curves were generated for the four sugars using concentrations ranging from 0. Excellent linearity was obtained, with a coefficient of determination greater than R2=0. Both Orange Juice A and B were diluted with water to obtain 10-fold dilutions, respectively, and after filtering the solutions through a 0. Table 2 shows the content values for the respective sugars detected in the juices. The grape juice was diluted with water to obtain a 10-fold dilution, and after filtering the solution through a 0. High Performance Liquid Chromatography L481 Analysis of Sugars and Sugar Alcohols in Energy Drink by Prominence-i with Differential Refractive Index Detector Sugars and sugar alcohols display almost no ultraviolet absorption, and are therefore typically detected using a differential refractive index detector or evaporative light scattering detector. By using a ligand exchange column for sugar analysis, it is possible to distinguish among the different isomers based on the position of the hydroxyl group in the chair conformation of glucose and fructose for example. In other words, the hydroxyl group of the sugar and the metal ion of the stationary phase form a complex, making it possible to achieve separation due to the difference in the strength of the complex formation. Also, maintaining a column temperature of 80 °C suppresses sugar anomer separation and peak dispersion, thereby achieving good separation of adjacent peaks. The column oven, which can accommodate a 30 cm column and maintain temperature control up to 85 °C, therefore supports applications that require a long column. When generating the curves for the six components over a concentration range of 0. Energy drink A was diluted 10:1 with water, and energy B, 20:1 with water, and after each was filtered through a 0. Xylitol and sorbitol were detected in energy drink A, and glucose and fructose were detected in energy drink B.
The ascospores of Neurospora tetrasperma Spore dormancy and germination Almost all spores are dormant treatment quadriceps pain order 100 mg phenytoin with visa, in the sense that their rate of metabolism is low symptoms 8 days after conception phenytoin 100mg low cost. But they can be assigned to two broad categories in terms of their ability to germinate medicine you can take during pregnancy generic 100mg phenytoin fast delivery. They do not germinate readily when placed in conditions that are suitable for normal treatment xanax withdrawal quality 100 mg phenytoin, somatic growth (appropriate nutrients, temperature, moisture, pH, etc. Instead, some of them require a period of aging (postmaturation) before they will germinate, and others require a specific activation trigger such as a heat shock or chemical treatment. By contrast, nonsexual spores show exogenously imposed dormancy they remain dormant if the environment is unsuitable for growth, but they will germinate readily in response to the presence of nutrients such as glucose. The cell becomes hydrated, there is a marked increase in respiratory activity, followed by a progressive increase in the rates of protein and nucleic acid synthesis. An outgrowth (the germ tube) is then formed, and it either develops into a hypha or, in the case of some sexual spores, it produces an asexual. The spore has a very thick wall (w) and is contained in the outer wall (ow) of the oogonium (female reproductive cell). The arrows mark the positions of antheridia (male sex organs that fertilize the oogonium). Their dormancy cannot be explained in terms of a general permeability barrier, because they are permeable to radiolabeled oxygen, glucose, and water. Instead, their dormancy is linked to an inability to use their major storage reserve, trehalose, which is not metabolized during dormancy, but is metabolized immediately after activation. The enzyme trehalase, which cleaves trehalose to glucose, is found to be associated with the walls of the dormant spores, separated from its substrate. Activation somehow causes the enzyme to enter the cell, as one of the earliest detectable events in germination. Constitutive dormancy of some other spores has been linked to endogenous inhibitors. For example, uredospores of the cereal rust fungus Puccinia graminis contain methyl-cis-ferulate, and those of bean rust, Uromyces phaseoli, contain methyl-cis-3,4dimethoxycinnamate. Prolonged washing of spores can remove these inhibitors, and this perhaps occurs when the spores are bathed in a water film on a plant surface. At first sight it seems surprising that a spore adapted for dispersal should have an endogenous inhibitor. However, this might prevent the spores from germinating in a sporing pustule (they do not require exogenous nutrients for germination) and ensure that they germinate only after they have been dispersed. Ecological aspects of constitutive dormancy the behavior of constitutively dormant spores often has clear ecological relevance. For example, a characteristic assemblage of fungi occurs on the dung of herbivorous animals. Their spores are ingested with the herbage, are activated during passage through the gut, and are then deposited in the dung where they germinate to initiate a new phase of growth. The spores of many of these coprophilous (dung-loving) fungi can be activated in the laboratory by treatment at 37°C in acidic conditions, simulating the gut environment. Examples include the ascospores of Sordaria and Ascobolus, the sporangiospores of several Zygomycota, and basidiospores of the toadstool-producing fungi, Coprinus and Bolbitius. Heating to 60°C activates the spores of many thermophilic fungi of composts (Chapter 11). It also activates the ascospores of pyrophilous (fire-loving) fungi such as Neurospora tetrasperma which grows on burnt ground or charred plant remains. Most of these fungi are saprotrophs of little economic importance, but one of them, Rhizina undulata (Ascomycota), causes the "group dying" disease of coniferous trees in Britain and elsewhere. It infects trees replanted into clear-felled forests, and the foci of infection correspond to the sites where the trash from the felled trees was stacked and burned. The ascospores are heat-activated around or beneath the fires, then the fungus grows as a saprotroph on the stumps and dead roots and produces mycelial cords that infect the newly planted trees. Once the cause of this disease had been recognized, the problem was easily solved by abandoning the practice of burning. However this is not possible in regions where lightninginduced fires are a periodic, natural occurrence. Some of the plants in these areas have become adapted to fire their seeds remain dormant for years until they are heat-activated. Some of the mycorrhizal fungi are similarly adapted, an example being the ascospores of the mycorrhizal Muciturbo spp. Basidiospores of the common cultivated mushroom, Agaricus bisporus, and of several mycorrhizal fungi (see below) show constitutive dormancy, but these spores will often germinate when placed next to growing colonies of the same fungus. The significance of this behavior could be to increase the gene pool of the colony. Dormancy and germination triggers in mycorrhizal successions Many forest trees of temperate and boreal regions. These mycorrhizal fungi produce a sheath around the root tips and extend into the soil as hyphae or mycelial cords (see. Several experimental studies have shown that a succession of mycorrhizal fungi occurs on young tree seedlings that are planted in forest nurseries or in previously treeless sites. Some of these fungi establish mycorrhizas rapidly on tree seedlings from airborne spores. Classic examples of these "early (pioneer) colonizers" are Laccaria and Hebeloma spp. Other mycorrhizal fungi are slow to establish in new sites, but colonize after several years and eventually become dominant on the root systems. These successional patterns have been studied in experimental field plots where the toadstools of mycorrhizal fungi appear above ground in autumn. These distribution patterns, in which mycorrhizas of "pioneer" or "early" fungi are found at the periphery of 400 300 200 100 0 25 50 75 Distance from base of tree (cm) 100 the root system, but are replaced by later colonizers (characteristic of older trees) near the tree base, were explained by two types of experiment. First, basidiospores were collected from fruitbodies of either "pioneer" or "later" mycorrhizal fungi and were added to pots of nonsterile soil. Only the pioneer fungi formed mycorrhizas in these conditions, and this was related to basidiospore germination, because only the pioneer fungi have basidiospores that germinate readily the spores of the "later" fungi germinate extremely poorly (often less than 0. Second, birch seedlings were raised aseptically (without mycorrhizas) then planted beneath older trees in a field site so that they would be infected by the fungi already established in the site. Some of these seedlings were planted directly into the undisturbed soil, but others were planted where the soil had first been removed with a corer (like the corers used for making holes in putting greens) and then replaced immediately. All the seedlings in the cored positions became infected by pioneer fungi, presumably from spores in the soil. By contrast, all the seedlings planted in undisturbed positions were infected by the "later" fungi, presumably from hyphal networks which radiated through the soil. So, the pattern of mycorrhizal establishment on birch in previously treeless sites can be summarized as follows. The pioneer fungi infect young seedlings in nurseries or in the field, from basidiospores that land on the soil surface and are washed into the root zone. They probably have annual cycles of infection from basidiospores as the root system grows and expands into new soil zones. The "later" fungi cannot establish initially because their spores germinate poorly. But they germinate eventually, especially in older parts of the root zone, and become dominant by spreading as mycelial networks to infect further root tips. In natural woodlands and forestry plantations, seedlings are likely to be infected directly by the "later" fungi, but in new sites they will initially be infected by pioneer fungi. This has practical consequences, because only the pioneer fungi are suitable for mycorrhizal inoculation programs, commonly used in land-reclamation sites. The fungi most often used for this are the puffball, Pisolithus tinctorius, and the toadstool-forming fungus Paxillus involutus. Both are pioneer colonizers that tolerate relatively high levels of toxic minerals and the low water-retention properties of mine-spoil and other land-reclamation sites. Exogenously imposed dormancy In laboratory conditions, most asexual spores germinate readily at suitable temperature, moisture, pH and oxygen levels. Some germinate even in distilled water, although most require at least a sugar source, and a few have multiple nutrient requirements. This is very common in soil (Lockwood 1977), and has also been reported on leaf surfaces. For example, spores often fail to germinate in topsoil, where the level of microbial activity is high, but they germinate in sterilized soil or in subsoils of low microbial activity. The germination that occurs in sterilized soil can be prevented if the soil has been recolonized by microorganisms, and even single microorganisms bacteria or fungi will restore the suppression.
Page 10 of 35 Technical Evaluation Report 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 Historic Use: Vitamins Status Livestock the existence and importance of vitamins treatment dvt buy discount phenytoin 100 mg on-line, a group of compounds considered essential to life medications like abilify generic phenytoin 100mg with amex, in various natural food products became understood toward the beginning of the 20th century symptoms 6 months pregnant generic phenytoin 100mg without prescription. Vitamin A was discovered between 1912-1914 medications like gabapentin discount phenytoin 100mg with mastercard, and the first synthesis of vitamin A was developed in 1947. Vitamin B2 was discovered in 1926, while other B vitamins niacin, folic acid, and vitamin B6 were discovered in the mid1930s. In 1747, naval surgeon James Lindin observed the importance of a nutrient contained within citrus fruits in preventing scurvy; Vitamin C was rediscovered in 1912 and was the first vitamin to be artificially synthesized in 1935. The causal relationship between vitamin D deficiency and incidence of rickets led to the discovery of vitamin D in 1922. In the same year, vitamin E was found as a component nutrient in green leafy vegetables (Obikoya, 2010). The addition of nutrients to specific foods can be an effective way of maintaining and improving the quality of the food supply, and a number of food products are fortified with vitamin compounds. As examples, dairy products are often fortified with vitamin D, while breakfast cereals and other grain products are commonly fortified with B vitamins. International Several international organizations have provided guidance on the fortification of feed for organic livestock production with synthetically produced vitamins. International organic regulations and standards concerning vitamins are described in the following subsections. Vitamins are included in the definition of feed additives and therefore subject to regulation. Specifically, the section of these guidelines pertaining to livestock production states that "feedstuffs of mineral origin, trace March 5, 2015 Page 11 of 35 Technical Evaluation Report 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 Vitamins Livestock minerals, vitamins, or provitamins can only be used if they are of natural origin. In case of shortage of these substances, or in exceptional circumstances, chemically well-defined analogic substances may be used" (Codex, 2013). In case these substances are unavailable, chemically well-defined analogic substances may be authorized for use in organic production. United Kingdom Soil Association Nature identical synthetic vitamins may be used in the production of non-herbivores without permission, while producers of herbivores must seek approval to use nature identical synthetic vitamins A, D and E. Soil Association standards do not permit the use of concentrated vitamins and minerals to encourage early maturity or high levels of production (Soil Association, 2014). Japan Ministry of Agriculture, Forestry, and Fisheries the Japan Ministry of Agriculture, Forestry, and Fisheries Standard for Organic Feed do not specify the allowed or prohibited status of vitamins in organic livestock feed materials. In case of a difficulty to obtain feed additives listed in 8, the use of similar agents to the described food additives are permitted only for supplementing nutrition and effective components in feeds. Thiamine mononitrate, vitamin A, vitamin B complex, vitamin B12 vitamin D3, choline chloride are present on List 4B, minimal risk other ingredients. Biotin, retinol acetate, riboflavin, nicotinic acid, pantothenic acid, vitamin E acetate appear on List 3, inerts of unknown toxicity. Evaluation Question #2: Describe the most prevalent processes used to manufacture or formulate the petitioned substance. Further, describe any chemical change that may occur during manufacture or formulation of the petitioned substance when this substance is extracted from naturally occurring plant, animal, or mineral sources (7 U. Individual vitamin compounds are produced on an industrial scale by chemical synthesis or partial synthesis, fermentation and/or by extraction from natural material sources. Selection of the manufacturing processes typically depends on available technology, cost of raw materials/chemical feedstocks, market prices and size, cost of implementing fermentation versus chemical processes (synthesis or extraction) and, to a lesser extent, the overall environmental impact of the production method. There is a high degree of structural diversity among individual vitamin compounds; as such, a large number of chemical reactions may be applied to the synthesis of vitamins. Chemical synthesis is advantageous for the commercial production of vitamins as it can be carried out in a continuous manner on an industrial scale. However, chemical synthetic processes can become increasingly complex when specific stereoisomers. For example, the chemical synthesis of myoinositol, an essential nutrient for many aquatic organisms, suffers from the difficulty of isolating it free of the other eight stereoisomeric forms (Henry, 1996). While chemical synthesis remains the dominant industrial production method for many vitamins, an increasing number of fermentation processes are being developed for vitamin production (Festel, 2005). Fermentation is an enzymatic process whereby microorganisms convert natural carbon-based nutrients. Many recently developed fermentation methods for manufacturing vitamins utilize genetically engineered microorganisms, generating concerns over the use of these vitamin sources in organic food production (Roseboro, 2008). Proponents of fermentative processes cite production cost savings, reduction in waste and energy requirements, and the use of renewable resources. Extraction from natural sources is widely considering inefficient and low yielding, making this the least utilized method of vitamin production for use in animal feeds and human supplements (Survase, 2006). An extraction method is described below in the context of vitamin E (tocopherol) extraction from various vegetable oils. The following subsections summarize common manufacturing methods used for vitamins. Processes reviewed in this section are provided as examples, and should not be considered the sole manufacturing procedures used for vitamin compounds. A breakdown of the commonly used production technologies for a subset of vitamin compounds is presented below in Table 3. Technologies Used in the Production of Vitamins Vitamin Vitamin A Vitamin B1 March 5, 2015 Commercial Methods Chemical synthesis Chemical synthesis Other Methods Fermentation, Extraction Fermentation Page 13 of 35 Technical Evaluation Report Vitamin Vitamin B2 Vitamin B6 Vitamin B12 Vitamin C Vitamin D3 Vitamin E Vitamin K Biotin Folic acid Niacin Pantothenic acid 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 Source: Festel, 2005 Vitamin A Vitamins Commercial Methods Fermentation, Chemical synthesis Chemical synthesis Fermentation Chemical synthesis Chemical synthesis Extraction, Chemical Synthesis Chemical synthesis Chemical synthesis Chemical synthesis Chemical synthesis Chemical synthesis Other Methods N/A Fermentation N/A Fermentation Extraction N/A Extraction Fermentation Fermentation N/A Fermentation Livestock Vitamin A is produced via a step-wise synthetic procedure. A representative synthetic method involves the reaction of geranial and acetone in the presence of sodium ethoxide and ethanol. The reaction initially forms pseudolonone, which is subsequently transformed to ionone in the presence of boron trifluoride/acetic acid (Solomons, 2000). Two sequential Wittig reactions complete the commercial synthesis of vitamin A acetate (Pommer, 1977). Hoffmann-La Roche employed a related synthetic method for the industrial production of vitamin A (McMurry, 2011). A 2010 patent was filed for a vitamin A production process using biofermentation with algae or yeast that are genetically modified to enhance the production of geranylgeraniol and farnesol, potential starting materials in the syntheses of vitamins A and E (Maurina-Brunker, 2010). Vitamin B1 Commercial production involves a six-step synthetic procedure (Williams, 1936). Beginning with ethyl 3ethoxypropionate as the feedstock for vitamin B1 production, the synthetic reactions include (1) formylation using ethyl formate, (2) reaction with acetamidine hydrochloride leading to aminopyrimidine ring formation, (3) replacement of aminopyrimidine hydroxyl group with a chlorine atom (chlorination) using phosphorus(V) oxychloride, (4) replacement of the labile chlorine atom with an amino group using alcoholic ammonia, (5) ammonium salt formation using hydrobromic acid, (6) introduction of the thiazole ring using 4-methyl 5-hydroxyethyl thiazole. A search of the patent literature revealed two methods for vitamin B1 (thiamine) production by fermentative methods. The first patent describes the development of mutants of the genus Saccharomyces Meyen emend Reess (yeast) for synthesizing vitamin B1 from sugars and inorganic salts (Silhankova, 1980). A more recent invention provides a method for producing thiamine products using a microorganism of the genus Bacillus containing a mutation. Vitamin B2 As of 2000, chemical production still accounted for a major component of industrial riboflavin synthesis. Subsequent coupling with a phenyl diazonium halogenide provides an azo compound, which is used in a cyclocondensation with barbituric acid to give riboflavin. The final step eliminates aniline, and trace amounts of aniline are commonly found in chemically synthesized riboflavin products (Stahmann, 2000). Microbial processes are currently replacing chemical riboflavin production methods in industry. Naturally occurring overproducers of riboflavin include hemiascomycetes Ashbya gossypii (fungus) and Candida March 5, 2015 Page 14 of 35 Technical Evaluation Report 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 Vitamins Livestock famata (yeast). Patents describing the use of genetically engineered bacteria, which overexpress the genes of enzymes involved in vitamin B2 biosynthesis, have been known since the late 1990s. A more recent patent developed a mutant of Bacillus subtilis bearing proline anologue resistance, which resulted in decreased susceptibility of the organisms to osmotic dehydration and increased vitamin B2 production (Lee, 2006). One method for the generation of nicotinic acid involves the oxidation of 3-methylpyridine using nitric acid in air as the oxidizing agent (Friedrich, 1988). Alternatively, the electrochemical oxidation of pi-deficient N-heterocyclic precursor compounds was described as a facile method for the synthesis of niacin in the patent literature; specifically, the electro-oxidative synthesis of niacin from 3-methylpyridine (Toomey, 1993). Vitamin B5 Calcium pantothenate is the form of vitamin B5 commonly employed in vitamin supplements and the fortification of food products. The conventional synthesis of calcium pantothenate involves three sequential chemical operations. Reaction of isobutyraldehyde with formaldehyde and cyanide initially yields racemic pantoyl lactone.
References
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