Page 347 - Vitamin D and Cancer

334 B.W. Hollis

DiaSorin test. In fact, this is basically how the FDA approves new devices for 25(OH)
D assessment through the 510 K process since the DiaSorin RIA was the first device
approved in 1993. The alternative is that each LC/MS site establish their own refer-
ence range which will take years of clinical study since a normal Gaussian distribu-
tion is useless in establishing a normative 25(OH)D range. In fact, this “normalization”
of values is quite common between other 25(OH)D assays and DiaSorin testing as
recent articles demonstrate [51]. For instance, if a recently published LC/MS article
was used for diagnosis, the levels reported would have to be increased by 13% if the
DiaSorin reference range is to be used for clinical diagnosis [19].
Finally, clinical reference laboratories should simply use a single reference
range to report circulating 25(OH)D levels as does Labcorp, 32–100 ng/mL.
Compare this to the Mayo Clinic which reports four different “classes” of 25(OH)
D status. This type of reporting is confusing and should be discontinued.

15.5 Methods of 1,25(OH) D Quantitation
2

Of all the steroid hormones, 1,25(OH) D represented the most difficult challenge
2
to the analytical biochemist with respect to quantitation. 1,25(OH) D circulates
2
at picomole (pmol) levels. The development of a simple, rapid assay for this
compound has proven to be a daunting task.

15.5.1 Radioreceptor Assay

The first radioreceptor assay (RRA) for 1,25(OH) D was introduced in 1974 [52].
2
Although this initial assay was extremely cumbersome, it did provide invaluable
information with respect to Vitamin D homeostasis. This initial RRA required a
20 mL serum sample, which was extracted using Bligh-Dyer organics. The extract
had to be purified by three successive chromatographic systems, and chickens had
to be sacrificed and Vitamin D receptor (VDR) harvested from their intestines. By
1977, the volume requirement for this RRA had been reduced to a 5 mL sample and
sample pre-purification had been modified to include HPLC [53]. However, the
sample still had to be extracted using a modified Bligh-Dyer procedure and then
pre-purified on Sephadex LH-20. Chicken intestinal VDR was still utilized as a
binding agent.
A major advancement occurred in 1984 with the introduction of a radically new
concept for the RRA determination of circulating 1,25(OH) D [54]. This new RRA
2
utilized solid phase extraction of 1,25(OH) D from serum along with silica car-
2
tridge purification of 1,25(OH) D. As a result, the need for HPLC sample pre-
2
purification was eliminated. Also, this assay utilized VDR isolated from calf
thymus, which proved to be quite stable and thus had to be prepared only periodi-
cally. Further, the volume requirement was reduced to 1 mL of serum or plasma.
This assay opened the way for any laboratory to measure circulating 1,25(OH) D.
2
This procedure also resulted in the production of the first commercial kit for