Vitamin tracers, radioactive

Replacement of Metabolic Losses An alternative approach to determining vitamin requirements is to measure the loss from the body pool in a steady state. This requires estimation of the total body pool, and measurement of the fractional rate of loss from that pool, generally using radioactive or stable isotope tracers. Three problems can arise in such studies. 

An alternative approach to determining requirements is to measure the fractional rate of catabolism of the vitamin by use of a radioactive tracer, then determine the intake that would be required to maintain an appropriate level of liver reserves. As discussed in Section2.2.1.1, when the liver concentration rises above 70 /rmol per kg, there is increased activity of the microsomal oxidation of vitamin A and biliary excretion of retinol metabolites. The fractional catabolic rate is 0.5% per day assuming 50% efficiency of storage of dietary retinol, this gives a mean requirement of 6.7 /rg per kg of body weight and a reference intake of 650 to 700 /rg for adult men (Olson, 1987a). Reference intakes for vitamin A are shown in Table 2.4. 

After a tracer dose of radioactive phylloquinone, the label is rapidly accumulated in the liver, then lost from the body with turnover time of 1.5 days. Tbis suggests that there is rapid turnover and little storage of vitamin K. However, there maybe considerable enterohepatic recirculation of the conjugates excreted in the bile (Shearer et al., 1996 Olson et al., 2002). About 10% of the total liver vitamin K is normally present as the epoxide, which is formed by the vitamin K-dependent carboxylase and normally reduced back to tbe active vitamin (Section 5.3.1). 

MK biosynthesis by the OSB pathway has been elucidated on the basis of isotopic tracer experiments, isolation of mutants blocked in the various steps, isolation and identification of intermediates accumulated by the mutants, and by enzyme assays. Early isotopic tracer experiments with various bacteria established that methionine and prenyl PPi contribute to the methyl and prenyl substituents of the naphthoquinone. The early isotopic tracer studies and other work have been reviewed by Bentley and Meganathan. " In 1964, Cox and Gibson observed that [G- " C] shikimate was incorporated into both MK and ubiquinone by E. coli, thus providing the first evidence for the involvement of the shikimate pathway." Chemical degradation of the labeled isolated menaquinone (MK-8) showed that essentially all of the radioactivity was retained in the phthalic anhydride. It was concluded that the benzene ring of the naphthoquinone (sic) portion of vitamin K2 arises from shikimate in E. coli The authors further suggested that shikimate was first converted to chorismate before incorporation into MK. A more complete chemical degradation of the MK derived from... 

In our studies, we have administered tritium-labeled vitamin A in one of its two physiological plasma transport vehicles (associated with either retinol-binding protein or chylomicrons) so that tracer data can be extrapolated to the vitamin A compounds of interest (retinol, retinyl esters, and metabolites). To prepare pH]retinol in its plasma transport complex (Green and Green, 1990b), vitamin A-depleted rats are used as donors to maximize hepatic secretion of the labeled vitamin on acciunulated liver apoRBP. pH]Retinol or pH]retinyl acetate in an emulsion with Tween 40 is administered intravenously to donor rats and blood is harvested 100 min later when plasma radioactivity is maximal. Plasma is isolated and stored under a nitrogen atmosphere at 4°C plasma is used for in vivo studies within 23 days. 

Immunoassays, specific protein-binding assays, and radioisotope tests are sometimes used for the determination of water-soluble vitamins. These are the only feasible and practical methods for the quantification of certain vitamins in physiological samples. The principle of competitive protein binding (CPB) using labeled radioactive or fluorescent tracer is still routinely applied to quantification of serum vitamin... 

The vitamin B12 activity of a given sample may be determined by biological, chemical, microbiological or physical methods including radioactive tracer, spectrophotometric, and partition techniques. The nature of the sample, the concentration of activity, precision, and time required will usually determine the assaj of choice. 

A radioisotope dilution assay and an assay based on animal growth are the most reliable methods for the determination of vitamin B12 activity. The tracer technique is highly specific for cyanocobalamin or analogs which are convertible to cyanocobalamin. The assay is specific for cyanocobalamin if cyanide treatment of the sample is avoided total cobalamins convertible to cyanocobalamin are determined if a given sample is first treated with cyanide. The method consists in the addition of a known amount of pure [ Co]-cyanocobalamin. A series of selective extractions and adsorptions to remove interfering substances is completed and the radioactivity and color of the purified sample are measured. From these data, it is possible to calculate the amount of cobalamin present in the original sample. The isotope dilution method is accurate and precise and can be used for both relatively pure samples and for crude extracts of low potency. 

Molhn and Baker (1954, personal communication) tried antibiotics in five patients with pernicious anemia. Antibiotics alone were ineffective. Moreover, antibiotics did not liberate enough vitamin B12 from intestinal bacteria to be effective even if gastric juice in doses up to 1000 ml. a day or equivalent amounts of intrinsic factor concentrate were given. Mollin and Baker also found that antibiotics given to patients with pernicious anemia did not improve the absorption of vitamin B12, as measured by their radioactive tracer technique (see page 158). 

All studies of the mechanism of riboflavin synthesis employing radioactive tracers, and the isolation of possible precursors in the biogenesis of the vitamin, have been carried out with the highly flaviogenic ascomycetes, Aahbya goaaypii or Eremothedum aahbyii. 

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