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Field of the Invention
The present invention relates to methods and materials for assaying mammalian blood and tissue. More specifically it relates to methods and materials for determining the amounts of vitamin B12 and vitamin B12 analogues in human plasma.
Prior Art
For many years it has been recognized that the assay of the vitamin B12 level in humans is a valuable technique for diagnosing and subsequently treating certain diseases, such as for example, pernicious anaemia, post gastrectomy states, nutritional deficiencies, intestinal disorders, and others. Initially, vitamin B12 was assayed microbiologically using either Euglena gracilis or Lactobacillus leichmannii. More recently, radioisotope dilution (RID) assays for B12 have been utilized. Such radioisotope dilution assay techniques are well documented in the literature, see for example Lau, et . al . ( 1965 ) "Measurement of Serum B12 Levels Using Radio isotope Dilution and Coated Charcoal," BLOOD, 26 , 202, as modified by Raven (1968) "The Effect of Cyanide Serum and Other Factors on the Assay of Vitamin B12 by Radio-Isotope Method Using 57Co-B12, Intrinsic Factor and Coated Charcoal," GUYS HOSPITAL REPORTS, 117, 89; and (1969) "Improved Method for Measuring Vitamin B12 in Serum Using Intrinsic Factor,

57Co-B12 and Coated Charcoal," JOURNAL OF CLINICAL PATHOLOGY, 22, 205.
Such prior art radioisotope dilution assay of vitamin B12 generally includes the steps of freeing the endogenous B12 from its natural binding protein by boiling at a selected pH and then adding a measured amount of radioisotope 57Co-B12 and a limited amount of binding protein. All of the binding protein will be bound by some form of B12 since the amount of radioisotope B12 added is, by itself, sufficient to bind the small amount of protein. As both the natural B12 and the radioactive B12 compete to bind with the protein, the degree to which the radioactive count of the protein bound B12 was inhibited was thought to be indicative of the amount of natural B12 present in the sample undergoing testing.
More specifically, In the technique of Lau as modified by Raven, serum B12 Is separated from
binding protein in the plasma sample by boiling with 0.25N HC1. Radioisotope B12 is added to the reaction mixture and the B12 mixture is reacted with protein, normally in the form of a commercially available binder. Then the free or unbound B12 is separated from the protein bound B12 by
protein-coated charcoal and the radioactivity of the supernatant liquid containing the mixture of bound radioactive B12 and bound non-radioactive B12 counted for radioactivity The serum B12 concentration is then calculated from the count, often by comparison with a standard chart. Almost as soon as this technique began to be utilized it was recognized that the vitamin B12 measurements it provided were usually Inconsistent with the results obtained by other meas uring techniques for B12, such as the microbiological assays. Most often, the vitamin B12 assay obtained by radioisotope dilution techniques have been found to be high. Many theories have been advanced to explain the cause of the high vitamin B12 readings. However, it is believed that nowhere in the prior art is it recognized that there are substances in mammalian blood and tissue which react with certain non specific protein binders in the radioisotope dilution assay technique to provide an analysis of vitamin B12 which is apparently higher than the amount of B12 actually in the sample. Additionally, It is believed that nowhere in the prior art is it recognized that most common and commercial RID assay protein binders are not specific to vitamin B12, but that they are also capable of binding with the heretofore unknown B12 analogues and thus provide erroneous B12 assays.
As has already been indicated, in the standard radioisotope binding assay for vitamin B12, a known amount of radioactive vitamin B12 is mixed with a prepared to-be-tested sample. Then, a known, but extremely limited, amount of protein which Is capable of binding with both the natural and radioactive vitamin B12 is added to the mixture. Then, utilizing well known techniques, the radioactivity of the bound sample is compared, for example, with a standard curve to determine the amount of natural vitamin B12 present in the tested sample. Such standard curves are initially established for use in RID assay, for example, by measuring the amount of bound radioactive B12 in the presence of the same type and amount of protein binder, but with several differ-ent amounts of known non-radioactive B12.
It has now been discovered, for what is believed to be the first time, that mammalian blood and tissue contain materials other than vitamin B12 which couple with certain binding proteins which are commonly used in RID assays. For purposes of this specification and claims the non-vitamin B12 materials which are capable of binding with such proteins will be herein referred to as "vitamin B12 analogues,"
"B12 analogues" or simply as "analogues." They are referred to as analogues, not due to their chemical structure, which is not known with certainty, nor in the commonly accepted chemical sense of the word "analogue." Rather they are referred to as analogues due to their reactivity with the binding proteins commonly used In RID assays. As will be shown in more detail, hereinafter, there are other similar-ities which have been discovered between vitamin B12 and the newly discovered analogues which are present in mammalian blood and tissue.
After the presence of B12 analogues was discovered it was then determined that protein binders commonly present in RID assays were: (1) Non-specific in binding to only vitamin B12; and (2) reactive in binding with both vitamin B12 and B12 analogues; and (3) capable of reacting with both B12 and B12 analogues independent of pH. These are most commonly R proteins. Additionally, it has been determined that other protein binders, are: (1) Very specific in their reactivity substantially only with vitamin B12;
(2) substantially non-reactive with the B12 analogues; and

(3) non-reactive with either vitamin B12 or B12 analogues In highly acid environments. These are most commonly
proteins in the form of pure human intrinsic factor (IF), hog IF, rabbit IF, other IFs and vitamin B12 specific
In the past the problem has been that RID binders Include substantial amounts of protein which is not specific to vitamin B12. Therefore, the radioisotope dilution
assay utilizing that binder on samples which contain B12 analogues will produce a measurement which indicates a greater amount of B12 present in the plasma than exists in fact. As will be shown in more detail hereinafter, commer cially available protein binders, which have heretofore been labeled as containing intrinsic factor, in fact include only about 10%. to about 30% intrinsic factor protein, while the balance of the protein in the binder is of a nonspecific type, such as R protein. Thus, the protein mater ials in the commercial protein binders are capable of
indiscriminate reaction with the heretofore unrecognized vitamin B-j 2 analogue materials in mammalian blood and tissue. These extraneous reactions give RID analyses having the appearance of apparently higher vitamin B12 content than the samples in fact contain. This is due to the fact that when the binder includes protein which is non-specific to vitamin B12 and which is capable of reacting with both vitamin B12 and B12 analogues, then the use of this protein in the radiobinding assay measures both the vitamin B12 and the vitamin B12 analogues which are present in the sample. How ever, In accordance with the present invention, when the proteins which are utilized are substantially specific to vitamin B12, such as substantially pure intrinsic factor, then in the RID assay one binds and measures substantially only the vitamin B12 in the sample, without the measurement of extraneous B12 analogues. This provides 'a more accurate vitamin B12 RID assay.
Based on these discoveries it is proposed that in the practice of RID assay only protein which is specific in its reaction to vitamin B12 be utilized. Alternatively, it is proposed that mixtures of vitamin B12 specific and non-specific binding proteins be treated, for example, with an excess of material which will bind or inactivate only the non-specific binding proteins, such as vitamin B analogues, prior to Its use in RID assays, so that the non-specific protein will be substantially unavailable for reaction with any vitamin B12 or analogues in a sample when the RID assay is conducted. In yet another modification of the present invention, crude binder, including non-specific binding proteins, is subjected to proteolytlc enzyme treatment prior to utilization as a vitamin' B12 binder In RID assays. Such proteolytlc enzyme treatment destroys the binding ability of the non-specific proteins without destroying the binding ability of the proteins which are specific to vitamin B12 Utilizing the techniques of the present invention, the B12 analogues can by assayed by analyzing the amount of vitamin B12 present utilizing, for example, a vitamin B12 specific binder, then assaying the sample utilizing a nonspecific binder and determining the difference between the two assays as a measure of the amount of vitamin B12 ana logues present.
These and other techniques are readily determined, once, as taught for the first time by the present invention, the presence of B12 analogues in mammalian blood and tissue is recognized.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the
in the following examples and tables certain chemical components were utilized. For ease of communication they have been given shortened names in the text. The concordance between the "component" names and their actual compositions is as follows:
Components Actual Composition
A. Buffer 1.0M Tris (hydroxymethyl)
aminomethane-HCl pH 10.0 B . Albumin Bovine serum albumin, 2 mg
per ml in H20
C . Salt 0.15M NaCl
D . Boiled buffer (1 part) 0.5M sodium acetate- HC1 pH 4.5
( 1 part) 0.01M KPO4 pH 7.5,
0.15M NaCl
(2 parts) 50 μg per ml KCN
in 0.15M NaCl
The complete solution is heated
of 45 min. at 100°C.
E. Standard Solution D containing 100 pg
(100 pg/ml B12) per ml vitamin B12. The solution is heated for 45 min.
at 100°C after the vitamin B12
Is added. The concentration
of vitamin B12 in the stock
solution used to make compone
E Is determined by its light
absorbance at 278, 36l and 550
F. Standard Same as component E except that

( B12) the vitamin B,p concentration
Is pg/ml.
G. (57Co) B 12 1000 pg per ml of (57Co) B12,
(150-300 μCl/μg), in H20.
H. Binder Present in 0.01M Tris-HCl pH
8.2, containing 0.15M NaCl and
50 μg per ml bovine serum albumin.
Binders are diluted in this
solution to reach a concentra
tion of 700 pg per ml of vitamin
B12 binding ability.
Individual binders are as follows:
1) Human Intrinsic factor
(Human IF) - Human gastric juice contain
ing more than 95% intrinsic factor based on assays employing inhibition of vitamin B12 binding with anti-intrinsic factor anti bodies ( 95% inhibition) and cobinamide ( 5% inhibition).
2) Human R protein (Human R)- Human saliva containing more than 95% R protein based on assays employing inhibition of vitamin B12
binding with cobinamide
( 35% inhibition) and anti intrinsic factor antibodies
( 5% inhibition)
3) Hog intrinsic factor (Hog
IF) - This protein was purified from "Hog intrin
sic factor concentrate" by affinity chromatography on vitamin B12-Sepharose employing gradient elution with
guanidine-HCl followed by
gel filtration. The final preparation contained more than 95% intrinsic factor based on assays employing
inhibition of vitamin B12
binding with anti-intrinsic factor antibodies ( 95%
inhibition) and cobinamide
( 5% inhibition).
4) Hog R protein (Hog R) (Also designated in the scientific literature as Hog non
intrinsic factor concentrate) This protein was purified from "Hog intrinsic factor
concentrate" as described above in 3). The final preparation contained more
than 95% R protein based
on assays employing inhibition of vitamin B12 bin
ing with anti-intrinsic factor antibodies ( 5%
inhibition) and cobinamide
( 95% inhibition).
5) Rabbit intrinsic factor
(Rabbit IF) - An extract of rabbit gastric mucosa
containing more than 95%
intrinsic factor based on assays employing inhibiti
of vitamin B12 binding with anti-intrinsic factor anti bodies ( 95% inhibition) and cobinamide ( 5% inhibition) .
6) Hog intrinsic factor concen trate (Hog IFC) - A crude
extract of hog pyloric mucosa. It contained 25% Hog IF and
75% Hog R based on assays
employing inhibition of vitamin B12 binding with anti-intrinsic factor
antibodies (25% inhibition) and cobinamide (75% inhibition).
7) Hog IFC + Cobinamide - Hog
IFC containing the vitamin

B analogue cobinamide (ECN, OH] Cbi) in a molar
amount equal to 100 times
the total vitamin B12
binding ability, i.e. a 100
fold excess of cobinamide.
8) Hog IFC + CN-Cbl [bde-OH] - The same as Item 7) above
except that the analogue
added is CN-Cbl [bde-OH]
and Is present In a 1000
fold molar excess.
9) Hog IFC + [3,5,6-Me3BZA]
(CN,0H)Cba - The same as
Item 7) above except that
the analogue added is
C3,5,6-Me3BZA] (CN, 0H)Cba.
10) Digested Hog IEC - Hog IFC
incubated with bovine pancreatic trypsin (2 mg per
ml) and bovine pancreatic
chymotrypsin (2 mg per ml)
for 60 min. at 37°C
I. Charcoal A solution containing 25 mg per
ml neutral charcoal (Norit)
and 5 mg per ml bovine serum
albumin in H20.
J. unknown sample Samples containing unbound
vitamin B12 are diluted in
solution D (see above). Samples
containing bound vitamin B12,
such as serum, are prepared as
(1 part) sample
(1 part) 0.5M sodium acetate- HC1 pH 4.5
(2 parts) 50 μg per ml KCN In
0.15M NaCl The complete mixture is heate
at 100°C for 45 min. followed
by centrlfugation at 5000x g at
4°C for 20 min. The superna
tant Is removed and used for
Each of the RID assays referred to herein utilized the components referred to above. The method and order of utilizing the components Is that set forth in Table 1. That is, components A, B, C, etc. or the buffer, albumin, and salt, respectively, etc. were added in the order, from left to right, shown in Table 1. After the addition of 57Co-B12 the components are mixed thoroughly to mix both the naturally occurring B12 and the radioisotope B12 to make them compete and equally available to react with the binder. After the addition of the binder, H, the components were again mixed thoroughly, and then incubated for 45 minutes at about 37°C Charcoal was then added to the incubated mixture and the com ponents again mixed thoroughly and incubated for another 5 minutes at room temperature. This was followed by cen trifuging at 2000 x g at 4°C for 30 minutes. Then 1000 μl of the resulting supernatant liquid is pipetetted from the sample and a determination of the amount of 57Co-B12 present is made. The amount of 57Co-B12 is indicative of the amount of natural B, 0 in the tested sample, with lesser amounts of

57Co-B12 being indicative of greater amounts of natural vitamin B12 in the sample.
Calculations of vitamin B12, utilizing the datea obtained in the foregoing manner, is made as follows:

Calculation of data from radiobinding assay f.or plasma vitamin B12 assay as outlined in Table I
1) The values in tubes 3 and- 4, the "blank" tubes without binder are averaged and subtracted from all other tubes starting with tube 5.
2) The background radiation is subtracted from tubes 1 and
2 and these values are averaged.
3) Tubes 5 and 6 are averaged. This value should be at,

least 15% below the average value for tubes 1 and 2 to insure that all of the binder Is saturated in the
presence of ( 57Co) B12 alone.
4) Values for each tube beginning with tube 7 are divide
by the average of tubes 5 and 6 to give values for " %
trace binding."
5) Percent trace binding for tubes 7-14 are used to
obtain a standard curve. We plot % trace binding on
the ordinate of logit-log paper versus pg vitamin B12
on the log scale.
6) The amount of vitamin B12 in unknown samples is
determined by interpolation from the standard curve of data of % trace binding versus pg vitamin B12.
7) The standard curves for all of the various binders used are virtually indistinguishable and vary little from
day to day. Nevertheless, a complete standard curve is always obtained for every binder with each set of assays. Representative data obtained with the assays are pres
ent In Table II.
Evidence as to the Origin and Existence of Vitamin B12
Analogues In Mammalian Blood and Tissue
Once the problem of the prior art is recognized, that is, that there are vitamin B, p analogues present In mammal ian blood and tissue, it becomes a relatively simple matter to prove the existence and chemistry of such analogues.
is also appropriate to prove that the various steps of the RID assay do not cause the B12 analogues to be formed, for example, from vitamin B12
In one instance this has been most convincingly shown by obtaining pure crystalline vitamin B12, subjecting var ious known concentrations of it to the same conditions used to extract endogenous vitamin B12 from blood and tissue sam ples (boiling for 45 minutes in the same extraction solution) and then analyzing them by RID assay using several binding proteins, for example, in the form of human IF, hog IF, human R, hog R and hog IFC on different portions of the same extracted vitamin B12 samples.

Referring to Table II, it will be seen that when
various known amounts of pure vitamin B12, ranging from about 8 pg to about 800 pg were tested with various
protein binders, that in each instance, the percent of radioactive trace binding, or more accurately, the inhi bition of 57Co-B12 binding, observed was substantially the same for each, binder. It is thus seen, that regardless of which protein binder is utilized, the percent
binding, i.e. inhibition of the ( 57Co)-B12 is substan
tially the same. This is indicative of the fact that during preparation for RID assay the pure vitamin B12 was not converted to analogues of the type which have now been ob served In mammalian blood and tissue. It is also indicative of the fact that in the absence of interferring masking components in the samples, such as B12 analogues, any of the binding proteins can be utilized to provide substantially equally acurate RID assays of vitamin B12.
Furthermore, the data in Table II should be suitable as a standard in the determination of vitamin B12 by the same RID assay.
By comparison, when endogenous vitamin B12 was extracted from serum from 74 normal blood donors (37 women, 37 men, ages 17-61) and tested utilizing the same binding proteins with the exception of hog IFC which was not used, the results were quite different. In every case in which serum from normal donors was tested greater inhibition of 57Co-B12 and therefore greater apparent vitamin B12, was observed with assays employing, as the binder, human R or hog R than was observed with assays employing human IF or hog IF. The data on the 74 normal donors is- included in Table III. Other data concerning patients with diagnosed vitamin B12 deficiencies are present, and comparisons between the
normal donors and patients have also been made on Table III, and will be discussed in more detail hereinafter. Referring to Table III it is seen that the mean endogenous vitamin B12 RID assay levels, in terms of pg of vitamin B12 per of serum, are 548 and 542 for human R and hog R,
respectively, but only 298 and 36l for human IF and hog IF, respectively. This demonstrates that something is present in extracts of normal human serum which inhibits the vitamin 57Co-B12 binding ability of human R and hog R to a greater extent than it inhibits the binding ability of human IF and hog IF. Under current RID assay teeh niuqes the greater inhibition which is found using human

R and hog R is analyzed to indicate a higher vitamin B12 content. It is those substances, which have now been found to be present in human blood serum and which preferentially inhibit 57Co-B12 binding of human R and hog R, which have been herein denominated as "vitamin B12 analogues."
Chemical Nature and Properties of Vitamin B12 Analogues
The vitamin B12 analogues, which are herein for the first time identified as being present in mammalian blood and tissue, have been isolated by paper chromatography and compared with pure vitamin B12. Vitamin B12 and the so called "vitamin B12 analogues" were found to have the following properties in common: (1) Both were adsorbed tb charcoal and remained adsorbed when the charcoal was washed with 5% phenol; (2) Both w-ere eluted from charcoal when the charcoal was washed with 67% acetone; (3) Both were extracted from aqueous solution into phenol and remained in the phenol phase even when the phenol was washed repeatedly with water; (4) Both passed into the aqueous phase when the phenol layer was dissolved in an excess of diethyl ether; (5) Both eluted with similar apparent molecular weights (approximately 1356) during gel filtration on columns of Bio-Rad P-4 polyacrylamide; (6) Both were adsorbed to a column of Sepharose-2B agarose that contained covalently bound hog R protein and both remained bound when the column was washed with 0.1M glycine-NaOH pH 10.0, l.OM NaCl, and both were eluted from the Sepharose with either 85% phenol or 60% pyridine* Because of these similarities the newly discovered material is seen to be similar to vitamin B12 and is thus referred to as vitamin B12 analogue.

The chemical nature and structure of the newly
discovered vitamin B12 analogues which are now found to be present in mammalian blood and tissue is not known. An effort was made to compare them with chemically true forms of vitamin B12, sometimes referred to in the literature as analogues of vitamin B12, namely CN-B12, 0H-B12, adenosyl B12 and CH3-B12, already known to be present in serum and tissues. This was done by adding 500 pg of each of these four known forms of vitamin B12 to four different portions of the same human serum, in the dark. Prior to the
additions the serum contained 250 pg and 450 pg of vitamin B12 as assayed by RID using human IF and human R, respectively, thus exhibiting a difference of 200 pg. After addition of the materials to the serum, each was allowed to incubate in the dark for 15 minutes to allow binding of the added known forms of vitamin B12 to the binding proteins normally present in the serum. Then the serum, with the added forms of vitamin B,p was extracted utilizing standard conditions and the apparent amount of vitamin B12 assayed by RID utilizing both human R protein and human IF protein. Both the human R and human IF assays showed an increase in the apparent amount of vitamin B12 of about 500 pg.
However, the original difference observed between the values obtained with the human R protein and the human IF protein, i.e. 200 pg, did not change. If any of the added known forms of vitamin B12 in the human serum had been converted to the newly discovered analogues, then the assays would have shown an increase in the difference. This provides evidence that the newly discovered vitamin B12 analogues were not formed from any of the known endogenous forms of native viatime B12 during the extraction procedure.
Isolation of Vitamin B12 Analogue
The materials which are herein designated as "vitamin B12 analogues" and which have been found to preferentially inhibit R proteins in the vitamin B12 assays were substantially separated from endogenous vitamin B12 by the follow ing purification scheme. A trace amount of 150 pg 57Co-B12, was added to l800 ml. of freshly collected normal human plasma. The added 57Co-B12 was sufficiently small that

It did not interfere with subsequent RID assays. After Incubating at room temperature for 30 minutes the vitamin B12 was extracted and assayed under standard conditions. When human IF binder was utilized in the RID assay the ext was found to contain 1050 ng of vitamin B12, but when huma R protein was utilized as the binder it appeared to contain 2030 ng of vitamin B12, almost twice as much vitamin B12. The extract was then passed through a column of
Sepharose containing covalently bound hog R protein. The column retained greater than 99% of the 57Co-B12 as well as the endogenous vitamin B12 as assayed by RID with human IF or human R protein. After the column was washed with a variety of buffers and water the material was eluted with 60% pyrldlne, taken to dryness under vacuum, dissolved in water, and adsorbed onto charcoal.. The charcoal was washed with 5% phenol followed by Water and the mixture of vitamin B12 57Co-B12 and analogue B12 was eluted from the charcoal with 67% acetone. The material was again taken to dryness under vacuum, dissolved in water, and then separated utilizing 19 inch long Whatman 3MM paper for paper chromatography and a solvent system consisting of 800 ml sec-butanol, 8 ml glacial acetic acid, 6 mmol HCN and 400 ml water. The chromatography was performed in the descend ing manner for 30 hours at room temperature in an environment that inhibited evaporation of the solvent. The paper chromatogram was allowed to dry in a fune oven and divided into 38 one-half Inch fractions and numbered, with fractio 1 starting at the point of application and number 38 being at the lowest point on the chromatogram. Each one-half inch fraction was then incubated with 5 ml of water at 4°C for twelve hours to elute the vitamin B12, 57Co-B12 and

B12 analogues. The water was then removed and taken to dr ness under vacuum. Each dried fraction was then, dissolved in 2.5ml of water and assayed for 57Co-B12 and for vitamin B12 using a variety of binding proteins. The final
recovery of 57Co-B12 was 64 % . The apparent recoveries of vitamin B12 were 15% when using human IF in the assay and 66% when using human R in the assay. The results of the assays employing the 38 fractions obtained by paper chromatography are presented in Table IVA, IVB, and IVC. Similar data concerning paper chromatography of 57 Co-B1 and pure vitamin B12, for reference as a control, are presented In Table IVD. The data in these several chroma tography tables summarized for convenience in Table IVE reveals that the behavior of 57Co-B12 that was extracted from human plasma did not change its chromatographic behav lor, and thus was not altered during the standard extraction procedure or any of the purification steps. In a similar manner it is postulated that true vitamin B12 is not
altered in any of the purification or process steps of the assay.
Referring to the control chromatogram of Table IVD, it is seen that the several RID assays of pure vitamin B12 performed variously with human R protein, human IF protein and hog IFC gave substantially a single symmetrical peak of activity. In each instance greater than 95% of the vitamin B12 was found to be present in fraction 14 through 16. Similar results, as shown in Table IVB, were obtained from the paper chromatogram of the plasma extract when the binding protein was human IF, hog IF and rabbit IF. These data are an indication that these three IF binding proteins are substantially specific in their binding ability to vitamin B12, and substantially non-reactive with vitamin B12 analogues present In plasma.
Efforts were made to modify hog IFC, which is a commonly used binder in RID assays and which has been found to con-tain as much as 90% hog R protein and as little as 10% hog IF, by removing or inactivating the hog R. In several Instances the hog IFC was incubated with an excess amount of three chemically synthesized vitamin B12 analogues before it was utilized in the RID assay. Referring to Table IVC, it is seen that after this modification the chromatogram results obtained utilizing the modified hog IFC closely resemble the results obtained with substantially pure hog IF. It is therefore seen, that in the practice of the

present invention, mixtures of protein including both vitamin B12 specific binding protein and binding protein which is not specific to vitamin B12 can be modified by the addition of an excess amount of vitamin B12 analogue, by which process the analogue binds with the non-specific protein to render it substantially bound or inactive so that it is not available to react with vitamin B12 or
( 57Co) B12 present in samples undergoing RID tests. The amount of vitamin B12 analogue to be added to a mixture of specific and non-specific proteins in order to bind or inactivate the non-specific proteins may vary over a wide range, depending on both the proteins which are present and the vitamin B12 analogues which are utilized as the binding or inactivating material. Generally speaking, for the exam pies shown in Table IVC, cobinamide may be added in an amount equal to that required for complete binding of the non-specific protein, up to an amount as much as ten million times greater than the amount needed to bind the protein, with the preferred range being about ten to about ten thousand times in excess of that required for complete binding. CN-Cb (bde-OH), known as CoB-cyano-cobamic a,c,g-triamide may be utilized in an amount at least about ten times to about ten million times in excess of the amount required to bind with the non-specific protein, with an amount of about one hundred to about one hundred thousand times excess being preferred. The (3,5,6-Me3BZA)
(CN,0H)-cba, known as Co (3,5,6-trimethylbenzimidazole) cobamide should also be utilized in amounts from about one to ten million times in excess of the amount of non-specific protein, with an amount in the range of about ten to about ten thousand times excess being preferred. Suitable amounts of other vitamin B12 analogues may be utilized in a similar manner to bind or inactivate non-specific proteins present in mixtures with specific proteins in order to obtain a preparation of binding protein which will substantially bind only 57Co-B12, or the vitamin B12 naturally present in the samples being tested, and thus give a more accurate quantitative RID assay of vitamin
B12 in samples undergoing tests.
Again, referring to Table IVC, data on samples of hog IPC digested with trypsin and chymotrypsin are shown. These and other proteolytic enzymes are specific in their ability to substantially digest R proteins while leaving intrinsic factor proteins unaffected and available as substantially the only protein for binding 57Co-B12 and vitamin B12 in

RID assays. Other enzymes, including, for example, elastase may be utilized for the same purpose. The amount of the enzymes utilized is in the range of about 0.01 to about 100 milligrams per mililiter of protein treated, with a preferred amount being about 0.05 to about 40 milligrams per mililiter of protein. Utilizing this proteolytic enzyme digestion process a protein binder is provided which sub stantially binds only 57Co-B12 and vitamin B12 and is not affected by vitamin B12 analogues in the samples being tested and which therefore gives a more accurate RID assay than is obtained when utilizing the original mixture of hog IPC proteins which included non-specific proteins which would have been capable of reacting with the newly discovered vitamin B12 analogues in samples to give inaccura assays as to the amount of vitamin B12 in test samples.
Now, referring to Table IVA, when samples from the plasma extract chromatogram were assayed for vitamin B12 with human R, hog R and hog IPC, different results were obtained than when those samples were assayed with human IP, hog IP, rabbit IP or hog IPC treated with vitamin B12 analogues or hog IPC digested with proteolytlc enzymes.
In each case where human R, hog R or untreated hog IPC we utilized as the binding protein the tests gave the appear ance that more vitamin B12 was present in the chromatogram samples, especially in fraction 1 through 13 and 17 through 38. This observation, when taken with the above data, pro vides strong evidence that normal human plasma contains a number of vitamin B12 analogues that compete with 57Co-B12 in significant amounts, for binding to R protein. It als indicates that such activity on the part of the B12 analogues Is substantially absent when the binding protein utilized in the RID assay is substantially specific to vitamin B12.
It should also be noted, see Table IVA, that the
chromatogram data suggests that the lack of specificity of human R and hog R is unchanged when RID assays are performed at acid pH. This indicates that erroneous results will be obtained for the true vitamin B12 content of samples which contain vitamin B12 analogues when RID assays are performed at acid pH.
Using the same techniques and criteria described above it has been discovered that vitamin B12 analogues are not only present in serum obtained from human blood, but are also present In mammalian tissues in even higher concen trations than they are in blood. Vitamin B12 analogues extracted from mammalian tissues have been purified using the same schemes as described above. When analyzed utilizing paper chromatography, they exhibited similar mobilities to those of the vitamin B12 analogues observed in the samples extracted from blood serum. Since larger amounts of the vitamin B12 analogues are present in tissue, they can be observed visually as red or orange spots during paper chromatography. The absorption spectra of vitamin B12 analogues purified from tissue extracts have been de termlned and demonstrate that they are similar to, but distinct from, the absorption spectrum of true vitamin B12 These observations provide additional evidence that the materials in blood serum which preferentially react with R proteins and not Intrinsic factor proteins are in fact varieties of vitamin B12 analogues.
The newly discovered vitamin B12 analogues also differ from vitamin B12 in terms of their biological activity.
Thus, as shown in Table V, the serum vitamin B12 values obtained with Euglena gracilis for eleven patients diagnosed to have vitamin B12 deficiency were substantially similar to the results obtained by RID assay using human IP or hog IP as the binding protein. It is to be further noted, that all of the values obtained by either microbiologic assay or by assay using human IP or hog IF were substantially lower than the values obtained when the RID assay was carried out utilizing human R or hog R as the binding protein.
This indicates that the vitamin B12 analogues which have now been identified in mammalian blood and tissue do not possess vitamin B12 activity of the type which is required to promote the growth of Euglena gracilis.
Data was obtained on ten additional patients diagnosed to be vitamin B12 deficient and the total of 21 patients with vitamin B12 deficiency are shown in Table V. In each of the 21 patients the vitamin B12 values found when the RID assay was carried out utilizing human IF or hog IF were below the range of vitamin B. ? values found in a control group of 74 normal subjects. However, when the RID assay was carried out utilizing human R or hog R only about half of the 21 vitamin B12 deficient patients were found to assay below the range of normal subjects for vitamin B12 This indicates that where the newly found vitamin B12
analogues are present in the samples being tested, and the binding protein is not specific to vitamin B12, the resulting assays may suggest that a truly vitamin B12 deficient patient is not within the deficient range. This may lead to "delay of treatment of that patient for vitamin B12
deficiency. It also Indicates that the vitamin B12 ana-logues that have now been discovered lack the therapeutic or beneficial activity of vitamin B12 in the sense of being unable to prevent the hematologic and/or neurologic
abnormalities associated with vitamin B. ? deficiency.
There are many commercial RID assay type kits available for the assay of vitamin B12 in clinical laboratories.
Table VI sets forth an analysis of several such kits, and a comparison of the types of protein found in those kits with hog IF, hog R and hog IPC. By reference to Table VI, It is seen that the commercial kits appear to have only about 13% to about 35% intrinsic factor and from about to about 85% R.protein. It is therefore suspected, that the use of these kits will give substantially erroneous assays of the amount of vitamin B12 present in a sample

when the sample also includes vitamin B12 analogues, such as those newly discovered to exist in mammalian blood and tissues. It has also been determined that the effectiveness of intrinsic factor to bind vitamin B12 is somewhat pH dependent, with intrinsic factor losing about 10% of Its binding ability at a pH of about 4.1 and losing about 99% of its binding ability at a pH of about 1.9. Thus, to the extent that the commercial kits use a pH of about 4.1 during binding they would have about 10% less intrinsic factor than shown in Table VI. Those kits having a pH during binding of about 1.7 to about 1.9 would obtain substantially no binding from intrinsic factor.
While the invention has been particularly shown and described with reference to preferred embodiments thereof, It will be understood by those skilled in the art that the foregoing and other modifications or changes in form and details may be made therein without departing from the spirit and scope of the invention.
What is claimed is :