Cobalamins in Human Serum

Table 26.1 Comparison of four different commercial available methods for analysis of total serum cobalamin. The four methods represent different principles for analysis of cobalamin in human serum.

Measurement of cobalamin in serum was introduced more than 60 years ago and, for clinical use, a number of assays are currently available on automatic platforms. All these assays have been well-validated but, due to minor differences in the assays, the results obtained by different methods are not directly comparable. Because of this, it is not possible to use a common reference interval across methods. In addition, comparison of the results from the measurement of cobalamin in food items and on human samples is currently hampered by a lack of a golden standard for the measurement of cobalamin both in biological samples and in food items. This issue should be solved in order to relate an accurate estimate of the intake of cobalamin to the level of cobalamin in human serum and in order to compare the results obtained by different laboratories. 

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). 

Lildballe, D.L., Hardlei, T.F., Allen, L.H., and Nexo, E., 2009. High concentrations of haptocorrin interfere with routine measurement of cobalamins in human serum and milk. A problem and its solution. Clinical Chemistry and Laboratory Medicine. 47 182-187. 

Kerwar et al. 1971 Peirce el al. 1975). The chemical structure of cobalamin and its derivatives in human serum is shown in Figure 26.2. 

Figure 26.4 Automatic measurement of cobalamin (Cbl) in human serum samples on platforms design of the methods. All three methods employ protein binding assays using intrinsic factor (IF) but differ in the detailed assay design. See the main text for details. The figure is published with permission from Clinical Chemistry Laboratory Medicine (Lildballe et al. 2009).

Three Automated Platforms Reacts Differently Towards Changes in Sample Type. This section describes the design of assays for cobalamin from the three main players in automated cobalamin analysis on human serum (Abbott, Bayer Diagnostics and Roche) and demonstrates how these assays react if employed for the measurement of cobalamin in human milk. Human milk contains up to 200-fold more haptocorrin than serum and most of it is unsaturated with cobalamin. If haptocorrin is insufficiently denatured, it can interfere in the assays (Lildballe et al. 2009). 

We discovered these analytical problems when seeking to analyse the content of cobalamin in human milk (Lildballe et al. 2009). However, we encountered the same problem for serum samples with a high content of unsaturated haptocorrin (Lildballe et al. 2011). Once such a problem is realized, it is possible to... 

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. 

Analysis of cobalamin (also known as vitamin B12) in human serum samples is very common worldwide as a mean to determine the cobalamin status in patients suspected to be cobalamin deficient. 

In order to be able to make an accurate comparison between cobalamin intake and cobalamin status of a patient in the future, there is a need to analyse cobalamin present in food items and in human serum employing... 

To summarize, human serum contains low concentration of various active and inactive forms of cobalamins bound to carrier proteins. Thus, in the development of any method for the measurement of cobalamin, these aspects must be considered. 

Figure 26.2 Chemical structure of cobalamin. X denotes the groups coupled to the central cobalt atom in cobalamins isolated from human serum. The crystal structure was first solved by Hodgkin and co-workers (Hodgkin et al. 1956).

In order to follow the imprecision and the accuracy of the analytical procedure over time, control samples must be included in each analysis. The control samples should be representative of the samples to be analysed and have concentrations comparable to those of the samples. Thus, for measurement of cobalamin in serum one should use a pool of human serum with concentrations of cobalamin in the low, intermediate and high level of the measuring range. Several programmes exist for monitoring the continuous quality of analysis (Thorpe et al. 2007) and are not discussed here. 

Today, measurement of cobalamin in serum is the most widely used blood test to judge the cobalamin status in humans. Currently, it is debated whether measurement of the fraction of cobalamin available for the cells, holoTC, will prove a more useful marker. The first assays suitable for measurement of this active fraction of circulating cobalamin have already been introduced in the clinical setting. 

Methods for analysis of cobalamin in other sample types other than human serum are less well-validated and it is important to validate them before employing such assays. 

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. 

Peracchi M, Bamonti Catena F, Pomati M, De Franceschi M, and Scalabrino G (2001) Human cobalamin deficiency alterations in serum tumour necrosis factor-alpha and epidermal growth factor. European Journal of Haematology 67, 123-7. 

The measurement of holotranscobalamin II is potentially useftil as a specific marker of biologically available vitamin Bi2, because only cobalamin bound to Tell is specifically available for uptake by aU cells. Other methods have been described for the measurement of holotranscobalamin in serum, one using an immobilized monoclonal antibody to human transcobalamin, followed by measurement of released cobalamin by CPB, This method is currently available as a commercial kit. The other method uses magnetic beads coated with cobalamin to precipitate apotranscobalamin followed by measurement of the holotranscobalamin in the supernatant by ELISA. Though these methods are claimed to be precise and simple to perform, there remains doubt over the interpretation of the measured concentrations, and over their sensitivity and specificity in the diagnosis of vitamin B deficiency. ... 

Serum isolated from a blood sample allowed to coagulate is a convenient human sample to collect in a reproducible manner. Therefore, the majority of routine analyses of cobalamins are designed to be used on serum samples. Alternatively, plasma can be used. 

Microbiological estimations of Bi in urine have limited value. Ideally the urine should be frozen and estimations of Bn should be made without delay. The least fecal contamination of urine and any bacterial prolifera tion may lead to grossly inaccurate results. After oral administration relatively little vitamin B12 appears in the urine, and serum levels are a much better indication of amounts absorbed. Vitamin Bn tolerance tests based on determinations of urinary excretion of the vitamin after intramuscular injection are described by Estrada et al. (1954). E. L. Smith (1953) found that most of the radioactive material excreted in the urine by a human subject after an oral dose of labeled vitamin B12 was not present as microbiologically active cobalamin. A urinary excretion test with radioactive vitamin B12 is discussed on page 159. 

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