Riboflavin radioactive labeling

Fig 1. Effect of incubation meditun and excess hormone on L-T and L-T/, uptake by mouse neuroblastoma cells (NBATA3) After 45 min preincubation confluent cells were incubated at 37°C for 2 h with 10 pM labeled thyroid hormone in the absence or presence of lOpM unlabeled hormone. Wells measuring 28.2 cm contained 4 ml of culture medium (RPMI 1640) or Hank s balanced salt solution. After washing with phosphate buffered saline +0.1% serum albumin, the cells were harvested and their radioactivity measured. Mean SEM of 3 experiments performed in duplicate. Experiments were performed under diminished light to avoid spontaneous deiodination especially of T, in RPMI due to riboflavin and folic acid. 

The glycine formed should be as avmlable to E. aahbyii for flavin formation as that obtained from serine (76). This proposal has been borne out by the experiments of Goodwin and Horton 85a) in which the incubation of totally labeled threonine-C with E. ashbyii was foimd to lead to incorporation of radioactivity only into positions C-4a and C-9a of riboflavin, that is, into the carbons previously found to be contributed by ycine (70). 

Because of the slow rate of flavin synthesis by the crude enzyme system, it has been difficult to determine the origin of the four-cari)on unit which condenses with 6,7-dimethyl-8-ribityllumazine to yield the o-xylene ring of the flavin. Two possibilities are apparent, either of which is conristent with the data obtained with radioactive glucose (86) (see Table III and Fig. 4). (a) Acetoin and diacetyl arise from pyruvate in metabolic qrstems and either one will condense chemically with 6,7-dimethyl-8-ribitylluma-zine to yield riboflavin. However, addition of diacetyl to the enzyme system from A. gosaypii did not enhance the incorporation of label from 6,7-... 

Goodwin and Treble (78a) have reported that the addition of 3-hydroxy-2-butanone-l-C to growing cultures of E. ashbyii led to incorporation of radioactivity into ring A of riboflavin exclusively. One-half of the was in the methyl groups and none in carbons 6 and 7 of the o-xylene moiety. The labeled acetoin was incorporated more effectively than acetate-2-Cn into ring A. 

The distribution of radioactivity in tissues and intestinal contents one hour after administration of 8a-[S-(N-acetyl)-L-cysteinyl]-D-[l- C]riboflavin is summarized in Table 2. That at least the flavinyl moiety must be absorbed from the gastrointestinal tract was indicated by the rapid and sequential appearance of a fraction of in those tissues, such as liver, that similarly reflect the uptake of [2- C]riboflavin. The rather rapid appearance of in kidney, especially after i.p. injection, was also typical for flavin localization and preceded excretion of a considerable fraction of the label. The considerable radioactivity in the small intestine and its contents after i.p., as well as per os, administration reflects entero-hepatic circulation. Only traces of 002 were exhaled within even... 

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