Background: The concentration of thiamin diphosphate (TDP) in erythrocytes is a useful index of thiamin status. We describe an HPLC method for TDP and its results in patients at risk of thiamin deficiency. Methods: We used reversed-phase HPLC with postcolumn derivatization with alkaline potassium ferricyanide and fluorescence detection. Samples were deproteinized and injected directly onto a C18 column. TDP concentrations in erythrocytes were compared with those in whole blood. Reference intervals for erythrocyte TDP (n = 147; 79 males and 68 females; mean age, 54 years) and whole blood TDP (n = 124; 68 males and 56 females; mean age, 54 years) were determined in an apparently healthy population. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency. Results: The method was linear to at least 200 μg/L. The between-run CV was <8%. The lower limit of quantification for both whole blood and packed erythrocytes was 300 pg on column with a detection limit of 130 pg on column. Recovery of TDP from blood samples was >90%. TDP in erythrocytes correlated strongly with that in whole blood (r = 0.97). Reference intervals for erythrocyte and whole blood TDP were 280–590 ng/g hemoglobin and 275–675 ng/g hemoglobin, respectively. Of the 63 patients suspected of thiamin deficiency, 46 were normal by both TDP and activation tests, 13 were deficient by both tests, 1 was deficient by the activation test but had normal erythrocyte TDP concentrations, and 4 were normal by the activation test but had low TDP. Conclusions: The HPLC method is precise and yields results similar to the erythrocyte activation assay.

It is known that Escherichia coli K-12 is cryptic (Phn ؊ ) for utilization of methyl phosphonate (MePn) and that Phn ؉ variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn ؉ variants are present at the surprisingly high frequency of >10 ؊2 in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn ؉ revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a highfrequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn ؉ and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.Escherichia coli can use P III compounds such as phosphite and organophosphonates, e.g., methylphosphonate (MePn) and aminoethylphosphonate (AEPn) as P sources. Their metabolism involves the enzyme C-P lyase, which appears to have a relatively broad substrate specificity (21). Whereas the mechanism of C-P lyase is not well understood, much is known about a cluster of 17 contiguous phn genes in E. coli, required for utilization of P III compounds (5, 21). The phnGHIJK genes within this cluster are thought to encode the core components of C-P lyase, while phnF and phnO potentially encode regulatory proteins (5). Several phn genes appear to encode components of solute transporters, and it has been deduced that, among these, the phnCDE genes encode an ABC type transporter. In this transporter, phnC encodes the ABC permease component, phnD encodes the periplasmic binding protein, and phnE encodes the integral membrane component.An interesting feature of the genetics of phosphonate metabolism in E. coli is that the B strain can use phosphonates whereas the K-12 strain is cryptic despite containing the entire phn gene cluster (21). The genetic basis for this crypticity was investigated by Makino et al. (14) and traced to an 8-bp insertion in the coding region of the phnE gene in the K-12 strain relative to the B strain, causing truncation of the phnE product. They also observed that the 8-bp sequence is one element in the direct t...

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SUMMARY.Three different methods for the assay of carcinoembryonic antigen were investigated-radioimmunoassay, enzyme-linked immunoassay, and a commercial immunoradiometric assay. Two standard preparations of CEA were used: the 1st British Standard (73/601) and a commercial preparation obtained as a component of the IRMA kit.Serum samples were assayed by each method against each standard material. Significant differences between results were obtained when the different methods were compared. When using the two standards in the same assay system no significant differences were found between results. A possible explanation and the implications of these findings for the routine assay of CEA are discussed.