Turnover studies

In general the turnover is presented numerically as the time required to change or alter one-half of the molecules present, i.e. the half-time (t /g). The turnover of lipids in vivo must be approached by the use of isotopes. The general technique is to administer isotopically labeled precursor material to the animal, to allow sufficient time for the precursor to be incorporated into the lipid under study, and then to measure the rate of loss of the isotope from the lipid. A plot of the log of the per cent isotope remaining in the lipid vs time normally gives a linear plot 

Similar studies designed to measure the turnover of cerebrosides of brain tissue have been carried out by a number of investigators. Davison et al. (1959) employed serine-3-to label brain cerebrosides and computed the turnover rate to be near 200 days. On the other hand Hajra and Rad in (1964) administered acetate-and measured the rate of loss of radioactivity from the fatty acid moieties of the cerebrosides. In the latter experiments the half-time was near 42 days. When the hexose of the cerebrosides is labeled by means of glucose-1- C or galactose-1- C, the rate of depletion of the isotope indicated a half-time of near 40 days (Burton, et al, 19 ). These data indicate that cerebrosides turn over slowly in brain, at a rate near 40 days, as measured by the hexose and fatty acid moieties. The data derived from the serine experiment undoubtedly indicates that the sphingosine pool in brain is small and that this compound is reused in the synthesis of the sphingolipids. 

Recent data obtained by Burton (impublished) on the turnover of cerebrosides sulfates showing the persistence of radioactivity in the brain tissue cerebroside sulfate, when sulfate- S is the label, indicates that the sulfate pool is small and that considerable reuse of the isotopic sulfate occurs. In these same experiments, a more rapid turnover of the hexose moiety of the cerebroside sulfate occurred than of the sulfate moiety. In fact, the half-time for the rate of loss of radioactivity from the hexose unit of cerebroside sulfate was identical with the rate of loss of the carbohydrate unit from cerebroside. 

The net result of these turnover studies is to show that lipids in brain are a part of the dynamic biochemistry of the body — even though they may function primarily as structural components. These data strongly suggest that considerable caution must be exerted in the interpretation of isotopic experiments conducted in vivo, especially when complex compounds are being studied. It emphasizes the need to know more than the radioactivity in the lipid. Other perimeters, such as pool sizes, turnover rates of the pools themselves, permeability, cellular barriers (such as the blood-brain barrier) and other related factors must be determined before a full explanation of the isotope data can be made. 

A large part of the sphingolipids are present as glycolipids. Metabolic Pathway 2, shown below, indicates the known routes of formation of the major nucleotide monosaccharide intermediates i.e. UDPgal, UDPglc, UDPgalNAc and CMPneu- 

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Hevesy, G. (1947). Radioactive indicators in turnover studies. Advances in Enzmol. 7, 111-214. 

The resynthesized triglycerides invariably contain saturated and unsaturated fatty acids in an approximate 2 1 ratio. Typically, nearly all of the fatty acid content of adipose tissue can be accounted for by just six different types of molecule with palmitate (06 0) and oleate (08 1) together contributing over 75% of the total. Turnover studies suggest that for most people, much of the fat is metabolically relatively inert acting as depot with a long half-life, and only a smaller component of the stored fat being readily accessible. 

A single turnover study of the conversion of the heme-HO-1 complex to free biliverdin has elucidated the relative rates of the catalytic steps 129). This transient kinetic study indicates that the conversion of Fe heme to Fe verdoheme is biphasic. Electron transfer to the Fe -heme HO-1 complex occurred at a rate of 0.11 s at 4°C and 0.49 s at 25°C with a 0.1 1 ratio of NADPH-cytochrome P450 reductase to heme HO-l complex. Oxygen binding to the reduced iron was sufficiently rapid im-der the experimental conditions that the species actually monitored... 

A key role of the promoter is to moderate the concentration of free iodide. This has a strong influence on catalytic rate, as carbonylation of [MeIr(CO)2l3] is poisoned by iodide. The mechanism in Scheme 3.2 shows how loss of iodide from [MeIr(CO)2l3] is required to generate neutral species which lie on the main pathway for catalytic turnover. Studies on the stoichiometric reaction steps (see Section 3.3.2.1) show that the methyl migration in the neutral tricarbonyl [MeIr(CO)3l2] is greatly favored over the anionic resting state. The mechanism also explains the influence of [H2O] on the Ir speciation. The equilibrium in Eq. (3) means that concentrations of water and F are coupled. Thus, high [HjO] leads to high [I , and... 

Pheromone (sex attractant). Ether extract of the stem, produced equivocal effect on Aspiculuris tetraptera, female and male Dacus dorsalis, male Mediterranean fruit flies, and male and female melon flies " k Pheromone (signaling). Ether extract of the stem, produced equivocal effect on Aspiculuris tetraptera, female and male Dacus dorsalis, male Mediterranean fruit flies, and male and female melon flies " k Phospholipidemic effect. Oil, administered to phospholipids transfer protein knockout (PLTPO)-deficient mice, produced an increase of phospholipids and free cholesterol in the VLDL-LDL region of PLTPO mice. Accumulation of phospholipids and free cholesterol was dramatically increased in PLTPO/HLO mice compared to PLTPO mice. Turnover studies indicated that coconut oil was associated with delayed catabolism of phospholipids and phospho-lipids/free cholesterol-rich particles. Incubation of these particles with hepatocytes of coconut-fed mice produced a reduced removal of phospholipids and free cholesterol by SRBI, even though SRBI protein expression levels were unchanged . 

A Arduini, A Peschechera, S Dottori, AF Sciarroni, F Seraflni, M Calvani. High performance liquid chromatography of long-chain acylcamitine and phospholipids in fatty acid turnover studies. J Lipid Res 37 684-689, 1996. 

In experimental serum sickness, a fall in serum complement level occurs at the time immune complexes form and inflammatory lesions develop (D6). However, levels of complement do not always reflect activation or consumption by immune complexes. The rate of synthesis of complement proteins may be sufficient to replace the amount being consumed, and several of the complement components are so-called acute-phase reactants, i.e., their levels rise with inflammation. Thus, activation may occur despite normal or even elevated levels in the serum. Turnover studies provide more direct evidence of complement utilization but are technically cumbersome (K4). A simpler approach is the detection of split products of complement components, which provides direct evidence of complement activation, or the examination of effusions for evidence of complement depletion (H31, N7, P7). 

It is possible to block the ability of LDL to bind to B-100,E receptors in fibroblasts (M14) and liver (C9, K26, W18) by modifying the arginine residues of LDL with cyclohexanedione. In vivo turnover studies using cyclo-hexanedione-modified and unmodified LDL have suggested that about two-thirds of LDL removed from the circulation in normal man is taken up by B-100.E receptors, and one-third by another mechanism (S26). 

ENZYME OVER-REDUCTION AND SUBSTRATE INfflBITION MULTIPLE TURNOVER STUDIES... 

Rehder, H., Schafer, A. 1978. Nutrientr turnover studies in alpine ecosystems. IV. Communities of the central Alps and comparative survey. Oecologia 34 309-327. 

Hepatocellutar Disease. Most forms of acute or chronic hepatocellular disease, including acute viral hepatitis and cirrhosis with jaundice, are associated with decreased levels of Hp, possibly caused in part by altered estrogen metabolism. Increased red cell breakdown secondary to erythrocyte membrane lipid alterations may also play a role, although this has never been documented with turnover studies. In contrast, biliary obstruction is also associated with significant lipid alterations but with increased Hp levels, in the absence of severe hepatocellular disease. 

Olesen, H., Turnover studies with iodine-labelled gamma-macroglobulin and albumin. Scand. J. Clin. Lab. Invest. 15, 497-510 (1963). 

Sternberg J, Imbach A. 1967. Metabolic studies with seleniated compounds. II. Turnover studies with Se75-methionine in rats. Int J Appl Radiat Isot 18 557. 

Finally, many enzymes are kinetically complex, and have multiple steps that partially limit both k at and k t/KM. One approach is to use single-turnover studies to obtain the rate of the chemical step and the kinetic isotope effects by this noncompetitive technique. Several examples of single-turnover studies of enzymes that exhibit the characteristic of turmeling are in the literature [11, 12]. Alternatively, tools that allow microscopic rate constants to be calculated from observed rate constants can be applied. This approach has been documented for peptidylglycine-a-hydroxylating monooxygenase [13], and more recently, for dihydrofolate reductase [14]. 

SLO follows an ordered, bi-uni mechanism, in which linoleic acid (LA) binds and reacts prior to O2 encounter [8], which has permitted a variety of steady-state and single-turnover studies into chemistry on SLO. The kinetic mechanism can be divided into a reductive half-reaction, described by the rate constant kcat/ffM(LA), and an oxidative half-reaction described by the rate constant fecat/KM(02). On the reductive half-reaction, SLO binds LA (ki), then the Fe +-OH cofactor abstracts the pro-S hydrogen from C-11 of LA (k2), forming a substrate-derived radical inter-... 

The measurement of neurotransmitter turnover is popular because it provides the investigator with potential modes of regulation of the dynamic equilibrium that controls transmitter pools. It is also an important measurement in light of the observation that in many cases transmitter tissue levels are barely disturbed after various pharmacological interventions or following neuronal depolarization and transmitter release. The question then presents itself as to how tissue pool sizes of transmitter remain relatively stable when the neuron is at disequilibrium. Turnover studies are an attempt to answer this question. 

Much has been written about the favorable effects of androgenic-anabolic agents on calcium balance, for which they have been widely used. A recent report combined both calcium balance and radioactive calcium turnover studies (25). It again showed the favorable effects of the steroids, which these authors attributed to a decrease in resorption of bone. 

Increased synthesis of Cp in response to RA was subsequently confirmed and shown to be related to disease activity [33, 65-66, 72-74]. Patients with severe to moderately active RA had significantly (P < 0.05) accelerated daily Cp turnover rates [65]. The highest turnover rate was found for a patient with the highest Cp concentration and severely active RA. Moderately active RA was associated with lower concentrations of Cp and moderate turnover rates. These results indicated that increased daily turnover was accompanied by increased rate of synthesis and, in the steady rate, a corresponding increase in elimination rate [66]. Gamma-globulin turnover studies also revealed an accelerated synthesis of this protein in the majority of patients with RA [66]. 

As a follow-up to our collaborative study with Underwood s lab (Lewis et al., 1981), we carried out a 35-day in vivo turnover study in rats (n = 11) with marginal vitamin A status (liver vitamin A ranged from 100 to... 

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