Choline formation

Lipotrophic factors are those required for transportation of triacylglycerol from the liver to the adipose tissue for storage. These factors are those that cannot be synthesized from nonlipotrophic components of the diet. The major role of lipotrophic factors is the formation of phosphatidylcholine, which is critical in VLDL formation. One of the lipotrophic factors obviously would be choline, which can be incorporated into phosphatidylcholine. Two other lipotrophic factors are related to the potential de novo synthesis of choline. The first and foremost is methionine, which can be used to donate the methyl groups for choline formation in the absence of dietary choline, thus allowing lipids to be moved from liver to adipose tissue (Fig. 18.7). 

A second lipothrophic factor is betaine, which is effective because the transfer of at least one of its methyl groups to homocysteine is very efficient and can replenish methionine for choline formation. In the absence of sufficient lipotrophic factors, a fatty liver develops, and there is insufficient movement of fats either ingested or synthesized in the liver to the adipose tissue. As fats enter or are synthesized in the liver, they are repackaged or packaged as VLDLs to be moved out for transport from the blood to adipose tissue. The VLDLs contain protein, triacylglycerol, cholesterol, cholesterol esters, and phospholipids, especially phosphatidylcholine (lecithin). If one has either a protein deficiency or a lipotrophic factor deficiency, the movement of triacylglycerol s from the liver to adipose is ineffective and a fatty liver can develop. Choline can be present in the diet and need not be synthesized de novo. Phospholipid synthesis has been discussed previously (Chapter 15). 

The demethylation reactions so far outlined take us to dimethylglycine. Nothing concrete is known on the further demethylation of this compound. Since N -betaine has been shown to be converted to labeled glycine the demethylation must go to completion. Similarly the role of sarcosine in the proposed cycle is obscure. In feeding experiments sarcosine was found not to be an effective methyl donor for choline formation. " This also was observed for the synthesis of methionine from homocysteine with liver slices and homogenates. The oxidation of the sarcosine methyl to formaldehyde, on the other hand, is a very active process and sarcosine oxidase is very widely distributed. This reaction could serve as a pathway for the catabolism of surplus methyl groups in the body. 

Another good example was described by McFarlane and co-workers. In this case the authors develop a novel self-polymerised ionic liquid 0L) gel prepared at room temperature (RT), without light or heat or addition of initiator, using choline formate (CF), and 2-hydroxyethyl methacrylate (HEMA). (Winther-Jensen et al., 2009)... 

Enzyme-Catalyzed Reactions Enzymes are highly specific catalysts for biochemical reactions, with each enzyme showing a selectivity for a single reactant, or substrate. For example, acetylcholinesterase is an enzyme that catalyzes the decomposition of the neurotransmitter acetylcholine to choline and acetic acid. Many enzyme-substrate reactions follow a simple mechanism consisting of the initial formation of an enzyme-substrate complex, ES, which subsequently decomposes to form product, releasing the enzyme to react again. 

Detoxifica.tlon. Detoxification systems in the human body often involve reactions that utilize sulfur-containing compounds. For example, reactions in which sulfate esters of potentially toxic compounds are formed, rendering these less toxic or nontoxic, are common as are acetylation reactions involving acetyl—SCoA (45). Another important compound is. Vadenosylmethionine [29908-03-0] (SAM), the active form of methionine. SAM acts as a methylating agent, eg, in detoxification reactions such as the methylation of pyridine derivatives, and in the formation of choline (qv), creatine [60-27-5] carnitine [461-06-3] and epinephrine [329-65-7] (50). 

In nutrition, the most important function of choline appears to be the formation of lecithin (phosphatidylcholine) (2) and other cb oline-containing pho sphohpids. 

All Kir channels are tetrameric proteins (see Fig. 3) of one-pore/two-transmembrane (1P/2TM) domain subunits which equally contribute to the formation of highly selective K+ channels. Most Kir channels can be assembled in functional homotetramers while some require heteromeric assembly (see Fig. 3). For example, functional GIRK channels underlying DCAch (Acetyl-choline-activated) current in atria are heteromultimers of two members ofKir3 subfamily Kir3.1 andKir3.4. 

Figure 24-2. Biosynthesis of triaq/lglycerol and phospholipids. ( , Monoacylglycerol pathway (D, glycerol phosphate pathway.) Phosphatidylethanolamine may be formed from ethanolamine by a pathway similar to that shown for the formation of phosphatidylcholine from choline.

One type of fatty liver that has been smdied extensively in rats is due to a deficiency of choline, which has therefore been called a lipotropic factor. The antibiotic puromycin, ethionine (a-amino-y-mercaptobu-tyric acid), carbon tetrachloride, chloroform, phosphorus, lead, and arsenic all cause fatty liver and a marked reduction in concentration of VLDL in rats. Choline will not protect the organism against these agents but appears to aid in recovery. The action of carbon tetrachloride probably involves formation of free radicals... 

Figure 6.1 Synthesis and metabolism of acetylcholine. Choline is acetylated by reacting with acetyl-CoA in the presence of choline acetyltransferase to form acetylcholine (1). The acetylcholine binds to the anionic site of cholinesterase and reacts with the hydroxy group of serine on the esteratic site of the enzyme (2). The cholinesterase thus becomes acetylated and choline splits off to be taken back into the nerve terminal for further ACh synthesis (3). The acetylated enzyme is then rapidly hydrolised back to its active state with the formation of acetic acid (4)...

Acetylcholine is formed from acetyl CoA (produced as a byproduct of the citric acid and glycolytic pathways) and choline (component of membrane lipids) by the enzyme choline acetyltransferase (ChAT). Following release it is degraded in the extracellular space by the enzyme acetylcholinesterase (AChE) to acetate and choline. The formation of acetylcholine is limited by the intracellular concentration of choline, which is determined by the (re)uptake of choline into the nerve ending (Taylor Brown, 1994). 

Crilly, K.S., Tomono, M. and Kiss, Z. (1998) The choline kinase inhibitor hemicholinium-3 can inhibit mitogen-induced DNA synthesis independent of its effect on phosphocholine formation. Archives of Biochemistry and Biophysics 352, 137-143. 

Another observation on oxalate formation is that other a-keto acids, such as oxalosuccinic acid (74) and a-ketoglutaric acid (106) do not seem to yield oxalate directly but indirectly (123). This appears to be due to the fact that only oxaloacetic acid can function as an acetate donor. In this connection the intervention of Coenzyme A may be considered, since it is reported to function in the acetylation of sulfanilamide and choline (73) and recently was shown to take part in the enzymatic synthesis of citric acid. This concept may be illustrated as follows ... 

The postsynaptic membrane opposite release sites is also highly specialized, consisting of folds of plasma membrane containing a high density of nicotinic ACh receptors (nAChRs). Basal lamina matrix proteins are important for the formation and maintenance of the NMJ and are concentrated in the cleft. Acetylcholinesterase (AChE), an enzyme that hydrolyzes ACh to acetate and choline to inactivate the neurotransmitter, is associated with the basal lamina (see Ch. 11). 

Acetylcholine formation is limited by the intracellular concentration of choline, which is determined by active transport of choline into the nerve ending 192... 

The rate of production of DAG in the cell does not occur linearly with time, but rather it is biphasic. The first peak is rapid and transient and coincides with the formation of IP3 and the release of Ca2+ this DAG is therefore derived from the PI-PLC catalyzed hydrolysis of phosphatidylinositols [1]. There is then an extended period of enhanced DAG production that is now known to be derived from the more abundant phospholipid phosphatidylcholine (PC), which has a different composition of fatty acid side chains [9]. Although DAG may be generated directly from PC through the action of PC-PLC, it can also be formed indirectly from PC. In this pathway, PC is first hydrolyzed by PLD to give choline and phosphatidic acid, which is then converted to DAG by the action of a phos-phatidic acid phosphatase [10,11 ]. 

A more successful strategy for developing sensitive and facile assays to monitor PLCBc activity involves converting the phosphorylated headgroup into a colorimetric agent via a series of enzyme coupled reactions. For example, phosphatidylcholine hydrolysis can be easily monitored in a rapid and sensitive manner by enzymatically converting the phosphorylcholine product into a red dye through the sequential action of alkaline phosphatase, choline oxidase, and peroxidase [33]. This assay, in which 10 nmol of phosphorylcholine can be readily detected, may be executed in a 96-well format and has been utilized in deuterium isotope and solvent viscosity studies [34] and to evaluate inhibitors of PLCBc [33] and site-directed mutants of PLCBc [35,36]. 

Because the preceding chromogenic assay rely on choline quantitation, the hydrolysis of substrates with headgroups other than choline cannot be followed. To circumvent this problem, another useful protocol was devised whereby the phosphorylated headgroup produced by the PLCBc hydrolysis is treated with APase, and the inorganic phosphate (Pi) that is thus generated is quantitated by the formation of a blue complex with ammonium molybdate/ascorbic acid 5 nmol of phosphate may be easily detected. This assay, which may also be performed in a 96-well format, has been utilized to determine the kinetic parameters for the hydrolysis of a number of substrates by PLCBc [37,38]. 

The physiologic sequelae of biotin deficiency are almost unexplored. Severe skin lesions, especially seborrheic dermatitis and Leiner s disease (Erythroderma desquamativum or exfoliative dermatitis), were increased in young infants bom of mothers on a restricted diet low in eggs, livers, and other biotin-rich foods. After biotin administration the lesions healed. There are claims that excess biotin produces a fatty liver characterized by heightened cholesterol content. Choline has no effect in the prevention of biotin-fatty livers (G2, M2). In mice with transplanted tumors, both the tumors and the blood levels of biotin are below normal (R8). More recent studies established a protection with avidin, the biotin-binding fraction of egg white, against tumor formation (K4). More data along these lines are still needed for confirmation. 

Two of these systems were studied as models—the acetylation of choline in brain to give acetyl choline (Hebb, Nachmansohn), and of sulfanilamide (the active component in prontosil, Chapter 3) in liver (Lipmann). Sulfanilamide is rapidly inactivated by acetylation on the p-amino group and then excreted. Sulfanilamide is easily diazotized the diazonium salt formed can be coupled with N-( 1 -naphthyl)ethylenedi-amine dihydrochloride to give a pink derivative (Bratton and Marshall, 1939). This formed the basis for an elegant colorimetric assay. Only the free p-amino group reacts, so that as acetylation proceeded color formation diminished. 

ET), which catalyzes the formation of CDP-ethanolamine, and (iii) an ethanolaminephosphotransferase (EPT), which finally synthesizes PE from DAG and CDP-ethanolamine. As discussed rmder PC synthesis, four enzymes have been cloned that can phosphorylate ethanolamine, two of which preferentially use ethanolamine as a substrate, and two which are more specific for choline. Only one isoform of ET has been cloned, which contains two active sites, but seems to be not as strictly regulated compared to its counterpart CT (Bladergroen et al, 1999a). 

Figure 11.21 Outline of synthesis of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine. Note in the synthesis of phosphatidylinositol, the free base, inositol, is used directly. Inositol is produced in the phosphatase reactions that hydrolyse and inactivate the messenger molecule, inositol trisphosphate (IP3). This pathway recycles inositol, so that it is unlikely to be limiting for the formation of phosphatidylinositol bisphosphate (PIP )- This is important since inhibition of recycling is used to treat bipolar disease (mania) (Chapter 12, Figure 12.9). Full details of the pathway are presented in Appendix 11.5. Inositol, along with choline, is classified as a possible vitamin (Table 15.3).

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