Cobalamins analogues

Kl. Kolhouse, J. F., Kondo, H., Allen, N. C., Podell, E., and Allen, R. H., Cobalamin analogues are present in human plasma and can mask cobalamin deficiency because current radioisotope dilution assays are not specific for true cobalamin. N. Engl. J. Med. 299, 785-792 (1978). 

Haptocorrin carries both the active and inactive forms of the vitamin (Hardlei and Nexo 2009). Methyl-cobalamin is the most abundant active form of the cobalamins bound to haptocorrin (Nexo 1977). The nature of the inactive forms, the so-called analogues, remains to be clarified. Cobalamin-analogues cannot act as coenzymes in the cells. Therefore, care is usually taken to choose a method that measures the active forms of cobalamin without interference from the analogues. The distribution of cobalamins and analogues associated with the two binding proteins is shown in Figure 26.1. 

For most analytical processes, a measured signal of the sample is compared with the signals of calibrators in order to calculate the concentration present in the sample. An ideal calibrator contains a known concentration of the analyte and is by all means comparable to the sample to be analysed. As serum contains various protein bound cobalamins and cobalamin analogues in a very complex matrix, it is not possible to use an ideal calibrator when analysing cobalamin in serum. A suitable approximation is a solution of cyanocobalamin prepared as described in Box 26.1. For the final dilution of the calibrator, a buffer comparable to the one used for dilution of the samples to be analysed should be employed. 

Determining the Snbtypes of Cobalamin. Based on differences in retention times between different forms of cobalamins, reversed-phase high-performance liquid chromatography (HPLC) has been used to separate the active subtypes of cobalamin and also to separate cobalamin from cobalamin analogues (Jacobsen et al. 1982). 

The detection system in HPLC is often based on ultraviolet (UV) light measurement of the effluent. Since the concentration of cobalamins in human samples is very low, our laboratory has developed a sophisticated method to determine low-level concentrations of HPLC-separated cobalamins (Hardlei and Nexo 2009). After immunoprecipitation of haptocorrin or transcobalamin followed by a proteolytic extraction and reversed-phase HPLC in darkness (to avoid conversion of methyl- and adenosyl-cobalamin into hydroxo-cobalamin), the fractions are vacuum-dried, re-dissolved in buffer and the cobalamin in the fractions is measured either by employing a sensitive cobalamin specific assay or an assay recognizing also the cobalamin analogues capable of measuring levels as low as 4pmol/L. 

Mass Spectrometry. Different varieties of mass spectrometry have for several years been used to identify the chemical nature of cobalamins produced from microorganisms (Kumudha et al. 2010). The sensitivity of mass spectrometry is still insufficient for measurement of cobalamins in the picomolar range and this method is therefore of limited interest for analysis of human serum. If the sensitivity is improved, mass spectrometry is predicted to be a very strong tool for identification of the varieties of cobalamins and cobalamin analogues present in human serum. 

When analysing cobalamin in human serum or developing new method a number of issues need to be considered cobalamin in serum is bound to two different proteins and it is present in various chemical forms, including inactive forms (the so-called cobalamin analogues). 

Cobalamin analogues Cobalamin analogues are cobalamin-related molecules not able to act as coenzymes for the cobalamin dependent human enzymes. In serum, both cobalamin and cobalamin analogues are present. The protein transcobalamin can only bind active forms of cobalamin, whereas the protein haptocorrin can bind both cobalamins and analogues. 

Gimsing, P., and Beck, W.S., 1989, Cobalamin analogues in plasma. An in vitro phenomenon . Scandinavian Journal of Clinical Laboratory Investigation. 194(Suppl) 37-40. 

J van Kapel, LJM Spijkers, J Lindemans, J Abels. Improved distribution analysis of cobalamins and cobalamin analogues in human plasma in which the use of thiolblocking agents is a prerequisite. Clin Chim Acta 131 211-224, 1983. 

JF Kolhouse, RH Allen. Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues. J CUn Invest 60 1381-1392, 1977. 

H Kondo, JF Kolhouse, RH Allen. Presence of cobalamin analogues in animal tissues. Pioc Natl Acad Sci USA 77 817-821, 1980. 

Makarov, A., and Szpunar, J. (1999). Species-selective determirration of cobalamin analogues by reversed-phase HPLC with ICP-MS detection./. y4na/./It. Spectrom. 14(9), 1323. 

The reduction properties of the cobalamins also differ from the normal Co111 complexes in that they can be readily reduced to the Co1 state, e.g. as in the methylation reaction (9). The electrochemical properties of the cobalamins have recently been reviewed.151 Several reviews are also available concerning their biological activity, the mechanisms of reactions, and synthetic analogues.150,152-155... 

Interest in these ligands stems from a desire to synthesize improved analogues of the cobalamines 100). With this in mind, 2,6-diacetylpyridine was condensed with N,N,N- f ra(aminopropyl)amine in the presence of nickel(II) or eopper(II) to afford the complex of 105, Cobalt(II) and zinc(II) have also been employed as templating agents in the synthesis of 105 101). The reaction of the nickel(II) complex of 105 with acetone results in a dimeric complex, 106101), by a process that is well established for primary amines 102). 

The active LCo complexes indicated above can be used to test this theory. Porphyrins and phthalocya-nines have an O-shaped system which has a more extended -system than that in cobalamins, but it does not provide a substantial increase in reactivity. It should be noted that the hydrogen bonds of the cobaloxime catalysts are essentially as effective as 7r-bonds in continuing the effects of delocalization around the macrocyclic ring. This effect has been noted elsewhere.142 Catalyst 11 comprises an O-shaped -system. Replacement of one jr-bond with a a-bond in the analogue 13 significantly affects the catalytic properties since both complexes retain their O-shape with -conjugation. Additional replacement of "T-bonds with o-bonds leads to a complete loss of catalytic properties as chelates 13, 20, or 21 indicate. Chelate 22, cannot be a CCT catalyst because of the absence of interaction between the two jr-systems. Chelate 34 is an exception its molecular structure is similar to 21 and 13, but it catalyzes chain transfer with a measurable rate. A possible explanation of this phenomenon will be provided in section 3.7. 

In complete B12-analogues the constitution of the nucleotide base can vary and the known classes of nucleotide fimctionahties found are benzimidazoles, such as the 5,6-dimethylbenzimidazole (DMB) of the cobalamins, purines, such as adenine and 2-methyladenine found in pseudovitamin B12 (10) and factor A (11), respectively, and phenols, such as p-cresol found in dicyano-p-cresolylcobamide (52) [145]. Sewage sludge is a particular rich, classical source of corrinoids [23]. Recent studies on the corrinoids from anaerobic microorganisms have shown a wide range of purine bases and benzimidazoles [1,6,37]. 

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