Page 348 - Vitamin D and Cancer

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15 Assessment of Vitamin D Status in the 21 Century 335
1,25(OH) D measurement. This RRA was further simplified in 1986 by decreasing
2
the required chromatographic purification steps [55]. This major improvement has
recently become a citation classic [56].
As good as the calf thymus RRA for 1,25(OH) D was, it still possessed two
2
serious shortcomings. First, VDR had to be isolated from thymus glands. Second,
3
because the VDR is so specific for its ligand, only H-1,25(OH) D could be used
2 3
as a reporter, eliminating the use of a I or chemiluminescent reporter. This was a
125
major handicap, especially for the commercial laboratory.

15.5.2 Radioimmunoassay

In 1978, the first RIA for 1,25(OH) D was introduced [57]. Although it was an
2
advantage not to have to isolate the VDR as a binding agent, this RIA was relatively
nonspecific, so the cumbersome sample preparative steps were still required. Over
the next 18 years all RIAs developed for 1,25(OH) D suffered from the same short-
2
comings. In 1996, we developed the first significant advance in 1,25(OH) D quanti-
2
fication in a decade [58]. This RIA incorporated and I-reporter, as well as standards
125
in an equivalent serum matrix, so individual sample recoveries were no longer
required. The sample purification procedure is the same one previously used for the
rapid RRA procedure [55]. The assay has 100% cross-reactivity between 1,25(OH) D
2 2
and 1,25(OH) D and is FDA-approved for clinical diagnosis in humans.
2 3
Another I-based RIA for 1,25(OH) D is also commercially available from IDS
125
2
Ltd. The basis of this kit is a selective immunoextraction of 1,25(OH) D from serum
2
or plasma with a specific monoclonal antibody bound to a solid support. This antibody
is directed toward the 1a-hydroxylated A ring of 1,25(OH) D [59]. This assay
2
procedure has never been published in detail so critical evaluation is difficult. I concluded
that this immunoextraction procedure was highly specific for the 1-hydroxylated forms
of Vitamin D. However, I also believe that this procedure overestimates circulating
1,25(OH) D levels. Evidence of this overestimation is evident in a recent publication
2
which shows a correlation of circulating 25(OH)D and 1,25(OH) D at physiologic
2
levels [60] indicating that 25(OH)D may be interfering with the assay.
ELISAs for circulating 1,25(OH) D determinations do exist commercially from
2
Immunodiagnostik and IDS. However, their performance has never been published
in detail. Further, no automated platforms or LC/MS methods yet exist for the assay
of circulating 1,25(OH) D.
2
References

1. Haddad JG, Chyu K (1971) Competitive protein-binding radioassay for 25-hydroxy cholecal-
ciferol. J Clin Endocrinol Metab 33:992–995
2. Hollis BW (2005) Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency:
implications for establishing a new effective dietary intake recommendation for vitamin D.
J Nutr 135:317–322