the use of chicken serum for measurement of serum vitamin b12 concentration by radioisotope...

The Use of Chicken Serum for Measurement of Serum Vitamin B,, Concentration by Radioisotope Dilution:

Description of Method and Comparison with Microbiological Assay R e d ts

RALPH GREEN,* PETER A. N E W M A R K , t ARTURO M. MUssOS AND DAVID L. MOLLIN?

tDepartment of Haematology, St Bartholowrew’s Hospital and Medical Collegc, Lotidoti. arid *Department of Haematology, School .f Pathology, Sorrth African Itistitirte for Medical Research,

johatttiesbitrg, South Africa

(Received 4 October 1973 ; accepted for priblicatiori I I December 1973)

SUMMARY. A new method for the measurement of serum vitamin B,, concentration by radioisotope dilution is described in which chicken serum is used as the sourcc of vitamin B, 2-binding protein. The conditions for the assay were carefully selected on the basis of a thorough investigation of thc factors affecting the critical steps in the radioisotope dilution assay for vitamin BIZ. The assay obeys the principlc of radioisotope dilution, and gives reproducible results. The normal range for the method (400-1020 pg/ml) is higher than any previously reported normal range for a radioisotope method. Excellent discrimination was obtained between normal subjects and those with pernicious anaemia.

The serum vitamin B,, levels measured by the chicken serum radioisotope dilution assay in 596 subjects were compared with the results of either Eiiglerta gracilis or Lactobaci/l~ts leichmanriii microbiological assay. The chicken serum method consistently gave higher results than either microbiological assay, but thedifference was especially great in many post gastrectomy, pregnancy, polycythaemia Vera and cord sera. A definite cause for these differences was not established.

Numerous methods have been described in thelast decade for measuring theserumvitamin B I ~ concentration by radioisotope dilution assay (RID assay) following the original method of Barakat & Ekins (1961) and this continued quest for improvement reflects a widespread dissatisfactioii with previously described methods. Some authors have found that the RID assay gives higher results than microbiological assays (Raven et al, 1969, 1972; Shun1 et a/ , 1970; Wide & Killander, 1971) whereas others have obtained lower values in some sera (Lau et al, 1965). Other reports (e.g. Friedner et al, 1969; Tibbling, 1969) have claimed good agreement between RID assay and microbiological assay. Thew disagreements may arisc

Present address: Department of Haematology. Central Military Hospital, Buenor hires, Argentina. Correspondence : Dr R. Green, Department of Haematology. South African Institutc for Mcdicsl Research.

p.0. BOX 1038, Johannesburg, South Africa.

507

508 Ralph Green et a1

from inadequacies in some RID assays and Friedner et a1 (1969) and Frenkel et a1 (1970). in particular, have studied some of the potential errors that might occur.

This paper describes a RID assay using chicken serum, and reports our experience with the clinical use of this assay for the diagnosis of vitamin BIZ deficiency.

MATERIALS AND METHODS

Ckicketi Serum Radioisotope Dilution Assay Reagerits. All chemicals should be Analar grade and solutions are made or diluted with

glass-distilled, de-ionized water. 5 7 Co-cyatiocobalarnin. High specific activity (100-300 pCi/pg) 5 7 Co-cyanocobalamin

(s7Co-BIz) (obtainable from the Radiochemical Centre, Amersham) is freshly diluted to a vitamin BIZ concentration of 1 0 0 pg/ml. 0.5 ml of this solution (50 pg) is used in each assay tube.

Statrdard cyanocobalamin (Std BI2). A solution of cyanocobalamin containing 1000 p g / d (‘Cytamen’, Glaxo) is diluted to give standard solutions of 1000,500, zso and 125 pg/ml. For the assays reported in this paper, the concentration of the starting solution was checked spectrophotometrically by measuring the optical density at its Em,, (361 nm).

Na-Acetate/HCI extracting bufer. pH 4.0, 0.4 I. A solution of 0.4 M Na-acetate is titrated to pH 4 with 0.4 N HCI. KCN is added to give a cyanide concentration of 20 mg/l.

Clycine/NaOH/NaCl neictralizing bufm. pH 10.0.0.8 I. A 0.8 M solution of glycine is made up in 0.8 M NaCI. This is titrated to pH 10.0 with 0.8 N NaOH.

Saline. 0.9% (0.154 M) sodium chloride. Chicken serum (CS). Serum, separated from chicken blood which has been allowed to clot

at 37°C. is suitably diluted with saline, and its unsaturated vitamin B12-binding capacity (UB,,BC) is determined. This is done by modifying the method of Lau et a1 (1965) so that binding is carried out for 60 min at room temperature in the presence of equal volumes (0.5 ml) of both the extracting and neutralizing buffers and in a reaction volume (2 ml) which is equal to that used in the assay. Based on the UB12BC, the CS is then appropriately diluted so that 0.25 ml will bind between 40% and 60% of the 50 pg 57Co-BIZ present in the assay. The UBIzBC of undiluted CS used in the present studies varied between 180 000 and 500 ooo pg/ml depending on the source of the serum and the dilution necessary was therefore between I in 1500 and I in 5000. In experiments carried out at St Bartholomew’s Hospital (SBH) blood was obtained from birds of a Rhode Island x Light Sussex chicken colony (by courtesy of Glaxo Laboratories). In experiments carried out at the South African Institute for Medical Research (SAIMR) blood was obtained from F, hybrid broiler chickens bred from Cornish Game Cockerels and White Rock pullets. Stored at - 2ooC the UB,,BC of diluted CS remains unchanged for at least 4 mth; undiluted CS stored at - 20°C appears to be stable indefinitely (Newmark et al, 1973).

Protein-couted charcoal is made up according to Lau et a1 (1965). A solution of haemoglobin is prepared from washed red cells by hypotonic lysis and the concentration adjusted to 10 g/Im ml. This is then dispensed into 5 ml volumes and stored in tubes at - 2ooC. Before use, the contents ofa tube are diluted to 200 ml with water, and added to 200 ml ofan aqueous suspension of 10 g Norit A, neutral activated charcoal (Amend or Hopkin and Williams).

Measurement of Serum Vitamin B,z 509 Alternatively, acid washed, activated charcoal (BDH) may be used, but this must be coated with albumin (Raven e l a/, 1969). 10.0 ml 20% bovine serum albumin (Arniour) is diluted to 200 ml and mixed with 200 ml of an aqueous suspension of 10 g charcoal. Coated charcoal stored at 4’C may be used for I week after preparation.

Assay method. In a previous paper we established the great potentiality of CS as the source of vitamin Blz-binding protein for use in a RID assay (Newmark et al, 1973). The present assay was designed to exploit this potential. In addition, during development of the assay, several other modifications of previous techniques were made; they will be described and justified in the Results section.

General principle. Serum vitamin BIZ is extracted from its complex with protein by boiling at acid pH. A known amount of 5 7 C ~ - B l Z is then allowed to compete with the extracted BIZ for binding to a limited amount of vitamin BIZ-binding protein. Finally, bound and free portions of vitamin BIZ are separated. Measurement of the amount of 5 7 C ~ B 1 2 that is bound allows determination of the degree of radioisotope dilution, and hence the amount of serum vitamin B12 present, by reference to a curve constructed from a series of standards containing known amounts of cyanocobalamin.

Procedure. The assay protocol is shown in Table I. All tubes in the assay are set up in dupli- cate in 75 x IZ mm Pyrex test tubes. Solutions and sera may be added with a variety of automatic dispensing equipment; coated charcoal may be dispensed with an automatic sampler, with continuous stirring of the charcoal suspension using a magnetic stirrer. Extrac- tion is carried out by placing tubes in a boiling water bath for 15 min. During this stage, evaporation is prevented by capping tubes with spring-loaded, metal ‘Capotest’ covers (supplied by C. E. Payne Ltd, London SW4 oHS) or non-absorbent cotton wool plugs. Optimal and consistent binding is achieved by addition of the neutralizing buffer, which renders the pH alkaline (ca 9.3) and maintains high ionic strength, followed by incubation with CS for 60 min at room temperature without shaking the tubes (Newmark et al, 1973). After addition of the neutralizing buffer, the CS and the charcoal, the tube contents are thoroughly mixcd. (A mechanical mixer, e.g. Gallenkamp ‘Whirlimixer’, may be used.) After the addition of coated charcoal, tubes are centrifuged for 10 min at 1850 g and 2 ml supernatant, containing bound vitamin B,z is taken off for counting. This step can be facili- tated by the use of an automatic diluter which draws up and delivers the required volume together with a I in1 water wash between samples to prevent carry-over of radioactivity between samples. In the present study, samples were counted in a gamma spectrometer (Packard autogamma) for 10 min each, which allowed a maximum counting error of 57; on statistical grounds.

Charcoat supernatant controls. In tubes to which CS has been added, the radioaaivity present in the supernatant after treatment with coated charcoal, should represent only the 5 7 C ~ B , 2

bound to CS. However, a small proportion of unbound 5 7 C ~ B I z may not be removed by coated charcoal and it then becomes necessary to correct the supernatant counts for the unbound radioactivity (Lau et al, 1965). To allow for this in the present assay, supernatant control tubes are included both for the test serum and the standard tubes (see Table I). These control tubes do not receive CS, but are treated with coated charcoal. The counts in both samples and standards ate corrected by subtracting the counts in the appropriate supematant control tubes. In practice we have found that all sera give very similar controls (always

v,

U

0

TABLE

I. Assay

prot

ocol

(vo

lum

es g

iven

in m

l)

unkn

own

sam

ple

supernatant

cont

rol*

(s

ampl

e)

Stan

dard

cu

rve

Supe

rnat

ant

cont

rol

(sta

ndar

d)

Tot

al

coun

ts

0.5

0.5

0.5

0.5

0.5

~~

Test

se

rum

0.2

5

0.2

5

-

-

-

Salin

e

-

-

0.2

5 t

0.2

5

0.2

5

0.5

-

0.5

-

Neut

ra-

lizin

g bu

ffer

--

Dilu

ted

chick

en

smm

0.2

5

-

0.25

-

-

Salin

e

-

0.2

5

-

0.25

1.25

Prof

ein-

co

ated

char

roal

1.0

1.0

1.0

1.0

- -

A s

ingl

e sa

mpl

e su

pern

atan

t con

trol

pre

pare

d fr

om p

oole

d se

rum

may

be

used

to

corr

ect a

ll sa

mpl

es in

a p

artic

ular

bat

ch (s

ee M

etho

ds).

t In

addi

tion

to s

tand

ard tu

bes

cont

aini

ng 0

.25

ml

of s

tand

ard

vita

min

BIZ

(125, 2

50,

500,

10

00

pg/m

l) a

set

of

dupl

icat

e tubes c

onta

inin

g no

add

ed

stan

dard

vita

min

B,,

is in

clud

ed to

ser

ve a

s zer

o po

ints

of

the

stan

dard

cur

ve.

Measwemefit of Serum Vitamin B, , 511

than 2.5% of the total counts). It is therefore sufficient to include a single control, prepared &om pooled serum, for any particular batch of samples instead of the individual sample controls found to be necessary by Lau et a/ (1965) and Frenkel et al(rg70).

Calculation of results. Following the principle of radioisotope dilution, the bound radioactivity present in the supernatant diminishes in the presence of increasing amounts of non- radioactive vitamin B I 2 . Thc standard curve is constructed by plotting the ratio of total counts addedlcounts bound (after subtraction of controls), on the ordinate (y) against the amount of standard vitamin BI2 added, on the abscissa (x). Constructed in this way, the standard curve yields a straight line when the system obeys the principle of radioisotope dilution. A typical standard curve is shown in Fig I. This linear form facilitates the calculation of results. From the equation for a straight line, y = bx+a, the amount of vitamin B,, present iii an unknown test serum sample (x) may be calculated as: x = I/b (y- a) where b is the slope, a the y-axis intercept, and y the value of total counts/counts bound for that sample. Since 0.25 ml serum is used in the assay:

total counts counts bound

vitamin BI2 (pglml) = 4/b ( Alternatively, the aniount of vitamin B,, in an unknown sample (x) may be read directly

off the graph after total counts/counts bound (y) is calculated for that sample. This value is multiplied by 4 to give the concentration of vitamin B,, per ml serum.

For the results reported in this study a desktop computer (Olivetti Programma 101 or 203) was used to calculate the parameters of the best line of fit for points on the standard curve

FIG I . A typical standard curve. Tubes containing 0, 31.25,62.5, 125 and 250 pg standard vitamin BIZ are shown.

(regression line by the method of least squares) and a second programme was devised which then calculated serum vitamin B,, levels from the ratio of total C O ~ ~ B / C O U ~ B bound according to the formula given above.

By way of an internal check on each assay we routinely calculated two further figures


from the parameters of the equation for the straight-line standard curve. These were derived from theoretical calculations that, if the amount of binding remains constant in all tubes of the standard curve as is close to the case under the chosen conditions of our assay (Newmark el al, 1973). then the binding capacity is given by 1/6 (the reciprocal of the slope of the standard curve), and the amount of 5 7 C ~ B , Z is givcn by the ratio u/b (which is iiumerically equal to the intercept on the x-axis). The derivation of these values is given in the Appendix.

Microbiological vitanrin B , assay Eirglena grucilis. The method of Hutner et a1 (1956) using the ‘Z’ strain, and modified by

Anderson (1964, was used at SBH where the normal range is I ~ O + Z S pg/ml (Anderson,

Lacttobacillirs kichntatinii. The method of Rosenthal & Sarett (1952) modified by Spray (1955) was carried out at SAIMR where the normal range is 220-1076 pg/ml (Brandt & Metz, 1961).

1964a).

SUBJECTS STUDIED

A total of 596 sera from normal subjects and from subjects with a variety of haematological and other conditions were assayed by the CS RID method and by either of the two microbiological methods.

Nornral srrbjects. These consisted of 94 normal Caucasoid blood donors from the South African Blood Transfusion Service and 39 healthy laboratory staff at SBH.

Vitanrin B , Deficiency Pernicioris anaemia. 108 subjects with proven, untreated pernicious anaemia (30 from SBH,

78 from SAIMR). Vitamin B,,-deficient sera. 49 subjects with subnormal vitamin B, levels by microbiological

assay (excluding any with the selected conditions given below) associated with atrophic gastritis, Crohn’s disease, idiopathic steatorrhoea, veganism or selective vitamin B , mal- absorption (26 from SBH, 23 from SAIMR).

Selected Conditions These were selected on the grounds of previous reports that the conditions were associated

either with abnormalities in RID assay results or with abnormalities in their serum vitamin B, binding proteins.

Fofate-deficiency. 37 subjects with megaloblastic anaemia (all from SBH). Partial gatrectomy. 1 x 0 subjects (66 from SBH, 44 from SAIMR). Pregnancy. 108 subjects in second or third trimester (all from SAIMR). Pofycythuemia vera. 14 subjects (all from SBH). Cord blood. 37 samples (10 from SBH, 27 from SAIMR).

RESULTS Development of C S RID Assay

During development of the procedure for the CS RID assay several techniques, preferred

Measurement of Serum Vitamin B, 513 &om theoretical considerations or on the grounds of convenience, were adopted after being justified by the following experiments.

Eflcacy of extraction. Preliminary experiments showed that protein precipitation was su- perior to hydrolysis for releasing vitamin B,, from the native vitamin BIZ binding proteinsin human serum. The extracting buffer we adopted was the best of several that were tested. Its efficacy was examined by counting the radioactivity in the supernatant after the serum proteins had been precipitated by boiling in the assay procedure. For 15 sera a mean ofg7% of the standard 5 7 Co-B, was released by the extraction procedure (range 9198%). Further- more, more than 98% of the radioactivity in the supernatant could be removed with coated charcoal, indicating that the 57Co-BI, was not bound to any unprecipitated protein in the supernatant.

Stage ofaddition of57Co-B,, . In many RID assays, the radioactive vitamin B,, is added to specimens after the extraction step. W e have deemed it preferable to add the radioactive vitamin B,, bcfore extraction, on the grounds that the 57Co-BIz is then subject to the same procedure as the serum vitamin B,, before the stage of competitive binding between the two takes place. In order to establish whether the stage of addition of 57C0-B,, has any effect on the assay results, assays were carried out on 10 sera adding the radioactive vitamin BIZ either before or after extraction. Agreement between the results of the two procedures was excellent for each serum.

Efict ofpresence ofprotein precipitate. During the stage of boiling in our assay, serum proteins are denatured resulting in the formation of a precipitate. In previously described vitamin BIZ RID assays which use this method of extraction, the supernatant is taken 06 and the assay is performed in the absence of the protein precipitate. Because of the remarkably avid binding of vitamin B,, by CS (affinity constant (K") = 1.1 x IO~' M- ') (Newmark er al, 1973) it seemed unlikely that the presence of denatured protein would interfere with the binding stage. T o test this hypothesis, 11 sera of varying vitamin BIZ concentration were assayed in the presence or absence of the protein precipitate. There was good agreement for individual sera and an excellent overall correlation in the results obtained with the two methods (I = 0.995, P < O.OOI), which confirmed that it was unnecessary to include the time- consuming step of removing the supernatant after extraction.

Addition Ofseriini to the standard curve. We have reported previously that the addition of pooled normal human serum (NS) to standard curve tubes in the CS RID assay did not affect the CS vitamin B,, binding capacity and we therefore predicted that the omission of NS from standards would not influence assay results (Newmark et a/, 1973). An experiment to test this was carried out in which standard curves were set up either routinely or with the addition of 0.1 ml of serum from a post gastrectomy patient (especially chosen because of the wide discrepancy between cs RID assay and microbiological assay of its vitamin BI2 level; 530 pg/ml and 160 pg/ml respectively). At the same time five Serum samples of varying vitamin BIZ concentration were assayed with or without the addition of 0.1 ml of the same post gastrectomy serum. As in the previous report the slope of the standard cuve, and hence the CS vitamin B,, binding capacity, was unaffected by the presence of serum. The CO-

efkient of correlation ( r ) between sera assayed routinely or with added post gastrectomy was 0.997 (P<o.ooI) and individual results did not vary by more than the limits of

reproducibility of the assay.


Alternative binders. In order to test whether the novel use of CS as the vitamin BIZ binding protein had, per sc, any great influence on the results of RID assay, a few assays were carried out in which CS was replaced by an alternative source of vitamin BIZ binding protein. The two binders used were as follows:

(I) Chronic myelogenous leukaemia serum (CMLS) which had a UB,,BC of 17 300 pglml was used at a dilution of I in 150 in saline. CMLS gave a straight line standard curve in accordance with the principle of radioisotope dilution (as with CS) which was not influenced by the addition of denatured protein (cf. intrinsic factor).

(11) Intrinsic factor (IF) was a 4 q f l saline solution of hog intrinsic factor concentrate (Armour). Although a straight line standard curve was obtained using IF, the presence of additional protein was observed to increase the binding capacity of IF (i.e. to decrease the slope of the standard curve). For this reason, in the experiment using IF, either 0.1 ml pooled normal human serum (NS) or hydrolysed and diluted NS were added to each tube in the assay. The hydrolysed NS was prepared in the following manner: 1.0 ml 0.25 N HCI and 3.0 mlo.rj M NaCl were added to 1.0 ml NS; the mixture was boiled for 15 rnin, diluted to 50 ml and 0.1 ml of this was added to each tube in the assay.

The assay results obtained using CMLS or IF were similar to those obtained by the standard CS RID assay procedure for the majority of sera tested (Table 11).

TABLE 11. Comparison of results @g/ml) obtained when alternative binders replaced chick serum in the RID assay

Chick serum

Chronic myeioid leukaemia s m m

Intrinsic factor 0.1 ml NS*

567 490

I37 3 77 348 43 I 642 528 837 1 9 0

I220

Intrinsic factor 0.1 ml boiled, diluted NS*

522 437

1 1 1 0 86 372 283

Normal serum (NS) added to all tubes in the may.

Evaluation of the CS RID Assay

of samples and between different batches. Reproducibility ofthe method. The reproducibility of results was checked both within batches

Meusurement of Serum Vitamin B, SIS Ten assays were performed on each of three sera in the same batch and in addition nine

sera were assayed in 10 different batches. The results are shown in Table III. There was less variation within a batch than between batches for sera of similar vitamin B,, levels, and in both cases the coefficient of variation was inversely related to the serum vitamin BIZ concentration.

TABLE 111. Reproducibility of the chicken serum RJD assay within and between batches

Serum vifamin B i z kuel(pg/ml)* Coe&ient ofvariation (%) I ~~

Mean Range 1 Within batch

643483 2.4 238-282 136-163

960-1086

61 1-746 274-329 23 1-294

129-223 103-rso 51-104

798-909

197-260

Bcfween barrh

4-1 5.0 6.0 s.8 9.4 9.8

15.2 15.7 I 8.7

Each serum was assayed 10 times.

Recovery of added vitantin B, 2 . A number of experiments were carried out in order to assess vitamin B,, recovery. In a typical experiment, 0.25 ml of each of the standard cyanme balamin solutions used in the CS RID assay (31.25, 62.5, 125 and 250 pg vitamin BIZ) were added to aliquots of 0.25 ml of a normal serum (vitamin B, 470 pglml), a post gastreaomy Serum (vitamin BIZ 516 pg/ml) and a pernicious anaemia serum (vitamin B12 250 p g l d ) and to aliquots of 0.1 ml of a chronic myelogenous leukaemia serum (vitamin BIZ 2180 pg/ml). All the samples were then assayed and the recovery of the added cyanocobalamin was calculated as: % recovery = [(std+serum) BIZ pg-serum BIZ p g ] ~ W s t d Biz pg. The recoveries were usually between 80% and 120% of.the added cyanocobdamin. Mean recoveries for the four amounts ofcyanocobalamin added to each serum were: normal 98.5%, post gastrectomy 98.5%, pernicious anaemia 92.0% and chronic myelogellous leukaemia

ofdihrtions ofsera. Two sera of normal vitamin B, level (950 pg ld and 565 p d d ) were assayed undiluted and diluted I in 2, I in 4, I in 8 and I in 16 with saline. At no dilution did the observed results deviate from those predicted by more than the experimental error (Table IV). It is therefore permissible to assay dilutions of sera and, in practice, this was carried Out when a vitamin B , ~ level of greater than 1000 pglml was obtained for any (undiluted) serum specimen.

119.8%.

Ralph Green et a1

Dilution*

X I

X Z

x 4 x 8 x 16

Serum Vitamin BI2 lewel(pg/ml)

Observed Predicted Observed Predicted

950 (950) 565 (56s) 520 475 270 a80 210 240 145 140 135 I20 90 70 40 60 4s 35

Assay of a mixture of sera. Since it was possible that there was a substance or substances present in some sera which might affect either the microbiological or the CS RID assay to give falsely low or falsely high results in either of the assays, experiments were carried out in which the vitamin B,, concentrations of mixtures of two sera were measured by both methods. Serum samples from a normal subject, a patient with pernicious anaemia and a gastrectomized patient were assayed separately by the L. Ieichmannii and CS RID methods. The normal serum was also mixed with an equal volume of either the pernicious anaemia or the post gastrectomy serum, and the mixtures were assayed. Both methods gave rcsults for the mixtures which were in good agreement with the expected results based on the assay of each serum separately. The observed and expected vitamin B, levels for the serum mixtures using the L. leichmannii assay were 220 and 280 pg/ml (normal+ pernicious anaemia) and 400 and 460 pg/ml (normal+post gastrectomy). Using the CS RID assay the observcd and expected results were 345 and 390 pg/ml (normal+pernicious anaemia) and 570 and 630 p g / d (normal+ post gastrectomy).

Resttlts o fch ick Serum RID Assay Healthy subjects. The vitamin B I Z levels of sera from 94 normal blood donors assayed a t

SAIMR ranged from 410 to 1145 pg/ml. The levels of sera from 39 healthy laboratory staff assayed at SBH were similar, with results ranging from 420 to 960 pg/ml. Thc distribution of the combined results is shown in Fig 2. The mean and standard deviation (SD) of thc serum vitamin BIZ levels of the combined results were 709k 154 pg/ml. This gave a calculated normal range (to the nearest 10 pg) of 4oo-10~0 pg/ml with 95% confidencc limits (mean fz SD) or 250-1170 pg/ml with 99.7% confidence limits (mean+3 SD). Thcse rangcs, together with the individual results, are illustrated in Fig 3.

Pertricioirs anaemia (PA). The observed range of results for the 78 PA sera from SAIMR was 55-330 pg/ml; all the values fell below the lower of the 95% confidence limits for the normal range (400 pg/ml) and 73 of them fell below the 99.7% limit (250 pglml). At SBH thc observed range for 30 PA sera was 35-490 pg/ml with all but one value below the 95% limit and 27 values below the 99.7% limit. The individual results are illustrated in Fig 3. Vitamin BIZ deficient sera (with subnormal vitamin B, levels by microbiological assay-see

‘Subjects studied’). Of the 23 sera from SAIMR all had values below the 95% limit and six of

Measurement of Serum Vitamin B, , 517

40r I

30

E a a 20 L

6 z 10

0 Serum vitamin 8,, (pg/ml) by chicken serum RID assoy

FIG 2. Distribution of m u m vitamin B,* concentrations in 133 normals measured by the chicken serum RID assay. Vertical line represents mean value.

these fell below the 99.7% limit. The corresponding numbers for the 26 samples from SBH were 24 below the 95% limit, six of which were below the 99.7% limit; the other two values fell within the normal rangc. Individual results arc illustrated in Fig 3 .

Selected conditions. The means and observed ranges of the CS RID assay results, at both SAIMR and SBH, in groups of gastrectomized, pregnant and folate-deficient patients, in patients with polycythaemia Vera and in cord serum specimens are shown in Table V. As expected, the mean results in the latter two groups were higher than in normal sera, and in each of the other groups a certain number of sera had subnormal vitamin B, , levels by CS RID assay (Table V).

Comparisoti of Chick Serum RID Assay with Microbiological Assays All sera whose vitamin B, , levels were determined by CS RID assay also had vitamin B, ,

levels measured by a microbiological assay. At SAIMR the assay organism was L. leichtnannii; at SBH it was E. gracilis.

Normal, PA and vitamin B , def;cient sera. A comparison of the results obtained by radioisotopic and microbiological assay of these sera isshown in Fig4 (CS RID assay YS L. leichmatrriii at SAIMR) and Fig 5 (cs RID assay vs E. gracilis a t SBH). In each case the calculated regression line is shown together with the (45') line of identity. It can be seen that the CS RID assay gave results which were greater than those of E. gracilis in every case and greater than L. leichsarinii in all but a few sera with high-normal vitamin B, levels. There was an excellent overall correlation between the CS RID assay and either L. leichniannii (r = 0.961, P< 0.001) or E. gracilis (r = 0.953, P< o.oor). It is apparent, especially compared with E. gracilis (Fig j ) ,

that the difference between the two assays for PA sera was often less than for sera with higher vitamin B,, levels. However when all the vitamin B,, deficient sera were considered as a group, the mean difference/( f SD) between their E. gracilir and CS RID assay results (161 k 70 pglml) was not much less than that for normal sera (198k 79 pg/d) . At SAIMR the mean

0. CI m

&

SAIMR

0

SBH

...

I

Folate deficiency

Vitamin BIZ (pglml) No. below

Mean Observed range (<4m pglml) No.

I334 713-3180

37

nonrral

18

477-1582 0

197-1210 9 I I


1200-

1100-

I000 -

5 0 900- a E $ 8 0 0 -

700-

f 600-

500-

5

s 2

N

mi

/ /

/ /*

I I I I I I I I I I I 0 100 200 300 400 500 600 700 800 900 loo0 1 1 0 0 1200

Serum vitamin B,, (pqhl) by L. /e&hmunii assay

FIG 4. Correlation between the results of chicken serum RID assay and L. feirhmrmnii assay of serum vitamin B,z concentrations in normals and in vitamin B,* deficiency (excluding selected conditions as in text). Open circles are cases of proven PA. The broken line is the(4so) line ofidentity. The solid line is the calculated regression line ( y = O . ~ Z S X + 146; r = 0.961).

difference between the L. leichmannii and CS RID assay results for vitamin B, , deficient sera (I3Ok60 pg/ml) was slightly greater than that for normals (112k I03 pg/d) .

Comparison in selected conditions. As expected, the mean results and observed ranges of serum vitamin B,, levels in groups of gastrectomized, pregnant or folatedeficient patients, in patients with polycythaemia Vera and in cord serum specimens were not as high when determined by either microbiological assay (Table VI) as they had been by CS RID assay (Table V).

The difference between the radioisotopic and microbiological. assay results for each individual serum was calculated, and the sera in each selected group were subdivided according to whether they had normal or subnormal vitamin B, , levels by microbiological assay. The individual differences in each group were then compared with the ‘normal range’ (mean +_ 2 SD) for differences between the two assays in either normal or vitamin BIZ deficient sera (excluding the groups of selected conditions).

A substantial number of sera in most groups had a difference between the two assay results which fell above the relevant normal range. The percentage of sera in each group having a recorded difference above the relevant normal range is given in Table VII. The highest

~ a l p h Green et a1

SAIMR (L. leichmannii)

/

/ J. /

D '

SBH (E. gradis )

._ 'v '""I

N I m 4001 .-

0

or w "*/ /

I -7 / I * /

I O P / i 200 *:a ,' 100% p / /

&/ 0

I I I 1 I I I I I I I 0 100 200 300 400 500 600 700 800 900 1000 1100 1200

Serum vilornin B,, (w/mi) by E. grocilk OSSOY

FIG 5. Correlation between the results of chicken serum RID assay andE. gracilis assay of serum vitamin BIZ concentrations in normalsand in vitamin BL2 deficiency (excluding selected conditions as in tcxt). Open circles are c a w of proven PA. The broken line is the (45") line of identity. The solid line is the calculated regression line ( y = I.O~OX+ 158; r = 0.953).

Srra I No.

Post gasaectomy Pregnancy Cord Polycythaemia Folate deficiency

Vitamin Bt 2 (pg/ml) No. below normal

(-20 pg/inl)

I i

40

, O I

No.

66 - - I0

14 37

norinal

22

72-890 I0

I I I I I

Measurement of Serum Vitamin BIZ 521

SAlMR Sera Vitamin BIZ level by

microbiologicat assay % with &@erence No. above normal range

-- Normal 94 1.1

Vitamin B,, deficiency I01 2.0 Post gastrectorny Normal 33 39.4

Pregnancy Normal 68 29.4 Subnormal 40 67.5

Cord Normal 27 88.9 Subnormal -

Polycythaemia Vera Normal - Subnormal -

Subnormal -

Subnormal I1 63.6

Folate deficiency Normal -

SBH

% with difference No. above normal range

39 5.1 $6 1.8

44 15.9 22 27.3 - -

8 87.5

14 36.0

26 15.4

2 I00

-

I 1 0

incidence was in cord sera and the lowest in folate-deficiency. In both pregnancy and post gastrectomy approximately twice as many sera with subnormal vitamin B I Z levels by micr- biologicai assay gave differences above the normal range as did sera with normal levels by microbiological assay. In post gastrectomy sera the incidence of high differences at SAIMR (L. leichmannii) was about twice that at SBH (E. gracilis).

DISCUSSION

The CS assay has a number of advantages compared with some other vitamin B, 2 RID assays. Firstly, as in the method of Lau er a/(1g65), it is not necessary to remove the protein precipitate after the extraction of vitamin B,, from serum. Secondly, as in the method of Raven et a1 (1969), a single serum supernatant control may be used for each batch. Thirdly, because the vitamin B,, binding capacity of CS is not influenced by the presence of denatured serum proteins, it is not necessary to add serum to the standard curve as it is when intrinsic factor is used as the vitamin B,, binder (Raven al, 19%; Hillman et al, 1969). Fourthly, the vitamin

binding capacity of CS remains virtually constant over the entire range of vitamin Bl 2

~ ~ ~ ~ ~ e n t r a t i o n ~ covered by the standard curve (Newmark rt a/, 1973) SO that the standard curve of our assay shows adherence to the form predicted from the basic principle of radioisotope dilution. Thus the standard Curve can be plotted in a linear form (Fig I) , which facilitates calculations, and it also allows a convenient internal check on the system (a/b = pg

CO-B,,, I/b = binding capacity of CS). Results ofthe chick seriitn RID assay. The normal range for serum vitamin B, concentrations

was 400-1020 pg/ml. Only Wide & Killander (1971) have previously reported a normal

5 7


range of this order (288-980 pg/ml) for a RID assay. There was very satisfactory separation betwcen serum vitamin B, , concentrations of normal subjects and those in paticnts with PA (Fig 3). Thus out of a total of 108 patients with proven PA only one had a serum vitamin B, concentration within the 95% confidence limits of the normal range and 100 (93%) had lcvcls bclow the 99.7% confidence limit normal range (250-1170 pg/ml). In practice, thcre- fore, a result below 250 p g / d was taken as evidence of an unequivocally low serum vitamin B,, level, with results between 250 and 400 p g / d constituting a ‘borderline’ range.

Cotriyarisoti qf the chick scrim RID assay with microbiological assays. Different methods of RID assay have given different normal ranges for the serum vitamin B, concentration, and these r~ormal ranges have not always corresponded with the normal range obtained using a microbiological assay even in the same laboratory. Discrepallt results between a RID assay and either the L. leichriiatitrii or E. gracilis microbiological assays have been described by a number of workers. With the exception of the report by Lau et al (1965) reported differcnces have always been associated with higher RID assay results (Shum et a/ , 1970; Widc & Killander, 1971; Raven el a / , 1972).

In preliminary communications we reported that our CS RID assay gave higher results than those ofmicrobiological assay (Muss0 ct al, 1971 ; Newmark eta / , 1972). This is confirmed in the present study where the CS RID assay results were consistently higher than those of either L. Zeichniannii at SAIMR or E. gracilic at SBH. Despite this, therc was an excellent overall correlation between the two assays (Figs 4 and 5). The finding that the difference between CS RID and L. Icichniatinii results tended to be less than that between CS RID and E. gracilis results is attributable to the fact that L. lcichniatinii at SAIMR gives higher results (normal range 220-1076 p g / d ) than does E. gracilis at SBH (normal range 160-925 pglml).

Our findings in pregnancy and post gastrectomy sera, but not in folate-deficiency sera, are in accord with the findings of others (Lawrence & Khpstein, 1967; Raven et a/, 1969, 1972; Hofirand, 1971). In these conditions the discrepancy in results between the CS RID assay and microbiological assay was particularly marked, and in a considcrablc proportion of the sera in these groups, differences between the two methods exceeded the calculated normal range of differences for either normal or vitamin B,, deficicnt sera (Table VII). Pregnancy and post gastrectomy sera which had subnormal vitamin €3, concentrations by microbiological assay displayed this marked difference more frequently than thosc with normal vitamin BI2 levels. This observation is of practical importance, since a number of pregnant and gastrectomized patients who might be regarded as vitamin B , 2 deficient from the result of microbiological assay had values which were within the normal range of the CS RID assay. Thus out of 40 pregnancy and 33 post gastrectomy sera which had subnormal vitamin B, levels measured microbiologically, 26 and nine respectively had normal RID assay results (Tables V and VI). Of possible relevance to this observation is the finding of Anderson (1964b) that a small proportion of post gastrectomy patients with subnormal vitamin BIZ levels by microbiological assay have only mildly reduced, or even normal vitamin B I Z levels in their livers.

The difference between RID and microbiological assay results vary considerably for individual sera but are not proportional to the vitamin B,, concentration as determined by either method. Moreover, these differences are more pronounced in certain conditions, SO

that the cause of the difference would seem to relate to an intrinsic property of particular


sera rather than to a systematic error in either method. Nonetheless, the differences between the assay results must be due either to an underestimation by the microbiological assay or an overestimation by the RID assay of the ‘true’ serum vitamin B,, concentration.

Overestimation by the RID assay could theoretically be related to a defect in the method. We have reasonably excluded the latter possibility by investigating each step in the assay, particularly the novel procedures in it, for a potential source of error. The stage of addition of ” Co-B, ,, the presence of precipitated serum proteins and the omission of serum from the standards were all shown to have no influence on the results of the CS RID assay. The use of CS as the source of vitamin B,, binding protein was also ruled out as a cause of the higher results, since essentially similar results were obtained when IF or CMLS was used in its place (Table 11).

Considering the other possibility, that microbiological assays give spuriously low results, our evidence provides no support for previous suggestions that either incomplete extraction of serum vitamin B,, (Raven et al, 1969) or an inhibitory substance (Lawrence & Klipstein, 1967) result in falsely low microbiological assays. Thus the finding of the predicted L. leichmannii results for mixtures of normal serum with post gastrectomy serum argues against the presence of an inhibitor and the similarity of the extraction techniques used for CS RID and L. leichmannii assays makes it unlikely that the technique for the latter is inadequate.

Nevertheless our findings do provide circumstantial evidence for one possible basis for the difference between RID and microbiological assay results. Although indicated for some years (Hall & Finkler, 1966), it has only recently been reported that, in addition to the two recognized vitamin B,, binding proteins in human serum, transcobalamins I and I1 (TCI and TCII), there is probably a third vitamin B,,-binding protein (Bloomfield & Scott, 1972). Like TCI, it is believcd to come from granulocytes and in fact it may simply be a TCI isoprotein (Carmel & Herbert, 1972). Raised levels of a third binder have been described in polycythaemia (Hall & Finkler, 1969), cord (Kumento, 1969) and pregnancy (Bloomfield i’t al, 1973) sera and it is noteworthy that in each of these groups we have observed a marked incidence of abnormally high differences between CS RID and microbiological assay results (Tablc VII).

APPENDIX

Derivation of the formulae I /b = binding capacity and a / b = pg 57C0-B12 added. Equatioii for the straight line is y = bx+ a, where b is the slope of the line and from Fig 6 is given by:

YI -Yo XI -xo

and a is the intercept on the y-axis (i.e. the value of y wheri x = 0). If the total counts are, say, k in ni pg 57Co-B12 added, and if the binding capacity of the added chicken serum is, say, N pg, then with no added standard B,, (xo), the counts bound will be:

(n /m) . k

t t /(ni+x,) * k and when s, pg standard B,, are added (x,), the counts bound will be:

Ralph Green et a1

FIG 6. A standard curve. At xo, no stairdard B I Z has been added. At x,. xl pg srandard B,, haw been addcd.

Since y is the ratio for (total coluits/bound counts)

Therefore

= m/n

yl = k/( 5 . k) m+ xI

= ( m + X , ) / J l

m + x , n1 XI

Y1-Yo = --- = - n 11 I 1

or n = XI/(Yl - y o )

Thus from (I), = I / b (3)

The binding capacity (n) is therefiregiven by the reciprocal ofthe slope of tht standmf ciirw Furthermore.

wheii x = o

&om (2)

y = a

= m / n

and

&om (3) a = m/(I /b )

m = alb

The amount ofs7C0-Bl, added (m) is thereforegiven by the ratio a /b Also from the equation for the straight line,

Measrrrenient of Serum Vitamin B,,

when y = o -x = alb

Therefore i n is numcrically equal to the intercept on the x-axis.

ACKNOWLEDGMENTS

The authors are grateful for the support given to them by thc Wellcome Trust (R.G.), the Medical Research Council (P.A.N.), the Universidad de Buenos Aires (A.M.M.) and thc World Health Organization (D.L.M.). They also wish to express their appreciation to Mr Nanu Pate] and Miss Sylvia Mestcr for their technical assistance, Dr Barbara B. Andcrson for helpful advice and supervision of the E. gracilis assay, and Miss Maureen Goodman for performing thc L. leichnramii assay. Pregnancy arid cord scra at SAIMR were studied in conjunction with Dr N. Colman. Dr Grccn is grateful to the Director, South African Institutc for Medical Rescarch, Medical Rescarch Council and Atomic Energy Board for the facilities to continue the work which he began at St Bartholomew's Hospital, and to Professor J. Mctz, in whosc Dcpartmcnt thc work was completed, for his helpful eiico~rag~rnc~it.

REFERENCES

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BLOOMFIELD, F.J., SCOTT, J.M., SOMERVILLE. J.J.F. 8: WEIR. D.G. (1973) Levels in normal, pathological and foetal r r a of the three tranxobalaniins. Irish Joirnml qf Medical Science, 142. 5 1 .

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526 Ralph Greerr et a1

LAWRENCE, C. & KLIPSTEIN, F.A. (1967) M e g a b blastic anemia of pregnancy in New York City. Annals of I n t e n d Medirine, 66.25.

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hvm, J.L., ROBSON, M.B., WALKER, P.L. Pr BARK- HAN, P. (1969) Improved method for measuring vitamin B I Z in serum using intrinsic factor, s7C0- BIZ, and coated charcoal. JorrrnalojCliniral Pafalltology, 23.205.

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the use of chicken serum for measurement of serum vitamin b12 concentration by radioisotope...

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