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Competitive protein-binding analysis of vitamin B12 using Lactobacillus leichmannii as a specific binder
155
Clinica Chimica Acta, 55 (1974) 155-163 @ Elsevier Scientific ~blishing Company, Amsterdam - Printed in The Netherlands
CCA 6465
COMPETITIVE PROTEIN-BINDING ANALYSIS OF VITAMIN Bit USING ~CTO~ACILLUS L~ICHMA~~II AS A SPECIFIC BINDER
D.F.M. VAN DE WIELa, J.A. DE VRIESb, M.G. WOLDRINGa and I-LO. NIEWEGC aCentml Isotope Laboratory, State University, Groningen, bPhilips-Duphar B. V., Weesp and ‘Division of Hematology, Department of Medicine, State University, Groningen (The Netherlands~ (Received March 3,1974)
Summary Vitamin B1 2 was assayed by the competitive protein binding method, using Lactobacillus leichmannii as binding agent. The intact bacteria were used as “solid phase”, and separation of bound and free vitamin B1 2 was obtained simply by filtration of the bacteria. Filtration could be rapidly done using a disposable syringe provided with a filter. Optimal conditions with respect to the extraction of vitamin B1 2 from serum and binding of the vitamin to the bacteria were determined. The effect of several drugs and hormones on the assayed vitamin B1 2 concentration was investigated. The correlation with the Lactobacillus leichmannii bioassay was calculated.
Introduction The growth rate of Lactobacillus leichmannii is the parameter commonly used in the microbiological assay of vitamin B1 2 {the bioassay). This communication describes an assay for vitamin Bt 2 which utilizes the method of saturation analysis, where La~tobacilZus leichmannii is employed as a specific binder . . for vitamin B, *. It was found by Kashket et al. [l], that the uptake of [ 5 ’ Co] vitamin B1 2 by the microorganism depends on the amount of nonradioactive vitamin B1 2 which is present in the incubation medium. This property makes it possible to determine vitamin B I 2 by means of competitive protein binding analysis, using the intact bacterium as “solid phase”. Separation of free from bound vitamin B1 2 can be done either by filtration or by centrifugation. McCall et al. [23 described this kind of assay for serum folate, and gave it the name of radiomicrobiological assay.
156
Materials and Methods
vitaminB1 *
standard solutions A stock solution was prepared by dissolving crystalline cyanocobalamin (Merck, Darmstadt, p.a.) in distilled water after which the concentration was determined by spectrophotometry. The solution was further diluted to a vitamin B1 2 concentration of 2000 pg per ml. This stock solution was kept in the dark at 4”. Standard solutions of O-1000 pg vitamin B1 2 /ml were freshly prepared every month by diluting 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml stock solution with distilled water to 20 ml. [5 ’ Co] Vitamin B1 z was obtained from Philips-Duphar B.V., Petten, The Netherlands, with a specific activity ranging from 100 to 300 mCi/mg. Purity and specific activity were examined by the method of Frenkel et al. [3]. The radiochemical purity was examined by the method of the British Pharmacopeia [ 41 and proved in all cases to be more than 90%. Before use the [’ 7 Co] vitamin B1 2 solution was diluted with water until the activity was 0.2 I.tCi/ml. 0.35 M acetate buffers 47.628 g CH3 COONa * 3Hz 0 (Merck, p.a.) was dissolved in 2 litres of distilled water. 1 litre of the solution was brought to pH 4.7 and 1 litre to pH 5.1 with concentrated HCl. Before each experiment 1 ml 1.0% KCN solution was added to 100 ml of each buffer. Lactobacillus leichmannii (American Type Culture Collection 7830) 0.2 ml of a suspension of Lactobacillus leichmannii (absorbance 0.600) was inoculated into 10 ml medium, which was a sterilised solution of 10 g glucose, 10 g yeast extract (N.S.G.F.), 1 ml Tween 80, 0.25 g thiomalic acid and 0.1 mg thymidinephosphate in 1 litre of water (final pH 5.3). The bacteria were incubated for 20 h at 37” after which the whole mixture was transferred to 1 litre of medium. After three days incubation at 37” the bacteria were washed with saline, and dried for 4 days at 37”. For use in the vitamin B1 2‘ assay, 10 mg bacteria were suspended in 10 ml 0.07 M phosphate buffer pH 7.0. The bacteria were centrifuged and resuspended several times, until the supematant remained clear. 1 ml of the stock suspension was mixed with 9 ml phosphate buffer pH 7.0, giving a bacteria content of about 0.1 mg/ml (dry weight). The suspensions were kept at 4” and were stable for at least three months. Serum ex traction 2 ml serum was mixed with 4 ml 0.35 M acetate buffer pH 4.7 containing 0.01% KCN in a glass tube, which was stoppered with a perforated plastic cap. The tube was kept in a boiling water bath for 15 min and then centrifuged (2500 rev./min). 3 ml of the supematant was pipetted into a plastic tube. Incubation 1 ml standard vitamin B1 2 solution (O-1000 pg/ml) was mixed - in duplicate - with 2 ml 0.35 M acetate buffer pH 5.1 containing 0.01% KCN in a
IO-* Fig. 1. Plastic disposable syringe, used for incubation (1 and 2) and filtration tube with incubation mixture: B: plastic adaptor with filter.
ml.Lact.1. (3)
-
of bacteria.
A: plastic
Fig. 2. Saturation curve of Lactobacillus leichmannii. 0.01-0.1 ml bacteria suspension (0.1 mg/ml) incubated with 30 pg [5 7Co] vitamin B12. All points were obtained from duplicate determinations.
was
plastic tube. To all standards and extracts 0.02 ml [’ 7 Co] vitamin B1 2 dilution (about 15 pg) was added plus 0.02 ml bacteria suspension (0.02 ml suspension binds about 50% of the [’ 7 Co] vitamin B1 2’). After mixing with a Vortexmixer the content of each tube was taken up in a plastic disposable syringe, and incubated for 120 min at room temperature. Separation of bound from free vitamin B1 2 During the incubation the syringes were provided with specially prepared filters (Selectron filter GF92, diameter 10 mm, C. Schleicher and Schiill, Dassel, G.F.R.) (see Fig. 1). After the incubation the mixtures were forced through the filters, and the filters were counted in a well-type gamma scintillation counter. Results The standard curve For the preparation of a standard curve it was necessary to determine the amount of bacteria that gave an optimal binding under the experimental conditions. This was done by making a “saturation curve” (Fig. 2). There appeared to be a reasonably good linearity up to a binding of 50%, which was obtained with 0.02 ml suspension. For this reason 0.02 ml suspension was used for the standard curve (Fig. 3). In one experiment filtration was compared with centrifugation. In the latter case 2 ml supematant was measured in a counter (Table I). The percentages bound after centrifugation were nearly the same, but in general filtration gave better results than centrifugation. For this reason and also because of its convenience we preferred filtration as the method of separation.
158
01.1, 0
200
400
600
&z(pg)
800
fOO0
-
Fig. 3. Standard curve with ~ctobac~l~us Zejc~~annii. 0.03 ml bacteria suspension was incubated with 30 pg [57Co]vitemin B12 and O-1000 pg unlabelled vitamin BI 2.
~~~~~~tio~ time, pH and temperut~re The incubation time was determined with the aid of several standard curves (O-1000 pg vitamin B1 2 ) which were obtained after various incubation ,times (Fig. 4). After 120 min the slope of the curve remained nearly constant, therefore 120 min was chosen as the incubation time. The influence of the pH on the binding was determined with 0.07 M acetate buffers (pH 3.5-6.0) and 0.07 M phosphate buffers (pH 5.0-8.0) (Fig. 5). It appeared that, in acetate buffer, the binding was strongly pH-dependent. Variation of the binding with temperature was determined by making three standard curves, at lo”, 21” and 35” with a fixed incubation time of 120 min (Fig. 6). Incubation at room temperature gave the best standard curve. Ex
trffction The method of extraction should meet the following requirements: (a) The serum proteins must precipitate qu~titatively, because of the
TABLE 1 SEPARATION TRATION
OF FREE AND BOUND
VITAMIN
B12 EITHER BY CENTRIFUGATION
OR BY FIL-
Vitamin BIZ @g) 1000
0
100
200
400
W Bound (t S.D., n = 2) after filtration
‘to.1 f 1.3
28.7 f 1.6
22.7 zk0.7
15.3 f 0.4
8.7 f 0.4
% Bound (t S.D., n = 2) after centrifugation
41.9 f 0.3
30.6 f 0.7
24.5 f 0.7
16.7 f 0.3
10.4 * 0.4
159
80
240 min //790 I) -130 I‘ -60 II \ 30 I,
20
t t
01 OO_L Blz(pg)
’
,
L
f
3.5 4
5
6
7
5
8
PH -
-
Fig. 4. Variation of the standard curve with the time of incubation. Fig. 5. Percentage bound as function of PH. lA,
acetate buffer; X -X
, phosphate buffer.
interference of soluble proteins with the binding between bacteria and vitamin 3, 2 (see below). (b) After centrifugation the volume of the supematant must be as large as possible because the sensitivity of the assay is such that much vitamin B1 2 is required. (c) The efficiency of the extraction must be reasonably constant, to avoid individual correction for each serum. (d) The extraction buffer must allow a good binding between bacteria and vitamin B, *.
0
1000
500 812 (pg)
-
Fig. 6. Variation of the standard curve with the temperature 0-e 10”: X-X 36O. . 21°;~------+
at a fixed incubation time of 120 min.
160
Buffers Two buffers were examined, glutamate [5], and acetate buffer according to Frenkel no precipitation of the serum proteins which the centrifugation step. The soluble protein, with the binding between vitamin B 1 2 and glutamate buffer was abandoned.
buffer according to Ceska et al. et al. [6]. Glu~a~ buffer gives offers the advantage of omitting however, appeared to interfere bacteria, and for that reason the
The su~ernatant uo~ume as function of pH and molarity 0.7 M phosphate buffers with pH 4.5, 5.0, 5.5, 6.0 and 7.0 were mixed with serum and treated as described under Materials and Methods (phosphate buffer was chosen at first, because the bacteria showed better B1 2 binding properties in phosphate than in acetate buffer). In addition several acetate buffers (pH 5.5) with molarities of 0.7, 0.35, 0.0’7 and 0,007 were mixed with serum and treated in the same way (Fig. 7). The largest supernatant was obtained with pH 4.5 and 0.35 M. Influence of molarity on the standard curve Three standard curves were prepared in potassium phosphate buffers (pH 7.0) which were saturated or with molarities of 0.7 and 0.07 (Fig. 8). There was a large difference between saturated and 0.7 M, but molarities between 0.7 and 0.07 did not appear to be critical. Efficiency of the extraction The efficiency of the extraction was determined by preincubating 2 ml serum with 30 pg [” ’ Co] stein B 1 2 for 30 min, followed by extraction as described under Materials and Methods. The supernatant was counted for radioactivity and the efficiency of the extraction, i.e. the percentage [’ 7 Co] vitamin B1 2 in the supematant, was calculated.
70-
30-
20 0
$1
* (1 300 812(P9)
Fig. 7. Volume of the supematant as a function of PH end molarity of the buffer. Fig. 8. Variation of the standard curve with the buffer molarity.
11 600 -
$1 900
161
Repeated extraction of one serum gave an extraction efficiency of 88.8 + 1.1 (S.D.). Extraction of six different sera gave an efficiency of 89.4 rt 2.9 (S.D.). The efficiency of the extraction appeared to depend on the ratio volume serum:volume buffer. In one serum this ratio was varied as 1: 1,1:3 and 1:7, giving an extraction efficiency of respectively 93.2, 98.2 and 99.2%. Consequently 0.35 M acetate buffer pH 4.7 was chosen as the extraction buffer because: (a) the supernatant volume was sufficient large, (b) a good standard curve could be obtained with this buffer, and (c) the extraction efficiency was reasonably high and constant.
The precision of the standard curve in the region of O-100 pgjml was found to be + 10 pg (SD.). The 95% confidence limit is therefore 20 pgjml, so that values below 20 pg/ml must be considered indistinguishable from zero (see also Discussion). Accuracy Pooled serum was diluted with distilled water to 80, 60,40 and 20% after which the vitamin B1 2 content was determined. In undiluted serum the vitamin Bi 2 content was 680 pg/ml. In the diluted sera the values found were respectively 540, 420, 300 and 220 pg/ml where as the calculated values were 540, 408, 300 and 136 pgfml. The accuracy was also tested by comparing the results with that of the “bioassay”. In the bioassay use is made of the fact that the growth of Lac~obac~f~us Ze~c~~a~~~~depends on the amount of vitamin B1 2 in the growth medium. Although in both assays, competitive protein-binding as well as bioassay, the same bacteria were used, the correlation between them was poor (Fig. 9). Precision A pool was made of 30 different sera, after which the vitamin B1 2 con-
800
x
t
i 600
(Ph
.
Z” a m,
0
(Y
400 i 200
i 0-b
0
. .
.
‘ii ii
l
l
t
l
.
l
..
. Q
.* b’
.
5
.
I
200 Radio
I
I
400
I
I
I
600
Bvz (Pg) m~crobioiog~cal
I
800
I
I
1000 assay
Fig. 9. Correietionbetween the competitive protein-biidiig assay and the bioassay. The equation of the regressionline wes y = 0.686x - 8.4. The correlationcoefficient was 0.756.
162 TABLE
II
PRECISION During
OF
the
are values
THE
seven
RADIOMICROBIOLOGICAL
days
obtained
between
from
duplicate
assay
ASSAY
I and
II the
sera
were
stored
at -2O’C.
Figures
Serum
Assay
N.
1000
I
Assay
665
(645.
685)
730
de G.
395
(390.
400)
380
Ha.
403
(375.
430)
330
O(
He.
brackets
II
970
Sch.
U.
between
determinations.
355
0. (355,
0)
20
355)
(
0.
40)
415
de V.
985
(970.1000)
R.
350
(300,
400)
350
D.
630
(575,
685)
675
P.
308
(305,
310)
340
A.
418
(390,
455)
430
K.
540
Sm.
395
(380,
410)
310
G.
590
(565,
615)
615
1000 (350.350)
515 (560,670)
tent was determined in twelve-fold: 379 f 45 pg/ml (S.D.). The vitamin B1 2 content of a series of sera was determined in duplicate. After storage of the sera at -20” during one week, the determination was repeated (Table II). Influence of drugs and hormones Several drugs and hormones were added to different aliquots of 2 ml serum, after which the vitamin B1 2 content was determined. The weight of the drugs was chosen such that it corresponded with a mean serum content after
TABLE
III
INFLUENCE
OF
DRUGS
AND
HORMONES
ON
THE
VITAMIN
B12
RADIOMICROBIOLOGICAL
ASSAY Added
per ml serum
Serum
0.0
495
0.4
mg tetracycline
HCI
0.1
mg barbital-
0.3
mg tolbutamide
525
1.0
mg sulfamerazine
490
Serum 420
500 520
4.0
~g nitrazepam
0.5
mg promazine
2.0
/lg acenocumarolum
535
2.0
mg salicylic
455
25.0
1 (pg B I z /ml)
525 HCl acid
ng oestradiol
515
440
6.0
ng testosterone
425
3.0
ng digoxine
412
1.0
/Ig progesterone
470
ng folic
485
30.0 0.1 10.0 1.4 107.0
acid
pg cortisol
417
l.(g diazepam
415
ng triiodothyronine
422
ng thyroxine
440
2 (pg BIZ
/ml)
163
maximal dosage, while the weight of the added hormones corresponded with the upper limit of the normal serum content (Table III). The values that were found all appeared to be within the range of precision. Discussion The sensitivity of the assay was not very high, therefore it was necessary to extract at least 2 ml serum. Several methods were tested to increase the sensitivity. First the manner of cultivat~g the bacteria was changed. ~~~~0~~~~~~~s ~e~c~~u~~~~is usually c~tiva~d in a vitamin 3, 2 ~ont~ning medium 191. To prevent a possible satumtion of the bacteria with vitamin B1 ?, thymidine phosphate [l] was substitu~d for vitamin B1 2. No large difference was found between “thymidine” and “Bl 2 ” grown bacteria: the decrease of the percentage bound between 0 and 1000 pg vitamin B1 2 was respectively 31.8 and 37.5. In addition another vitamin Bi 2-binding bacterium was tested, which is known to be able to grow on a vitamin B 1 z’-deficient medium, namely Lactobacillus casei [71. The sensitivity of Lactobacillus casei however was much less: the decrease of the percentage bound was 19.0. An advantage of the assay is the very rapid separation of free and bound . . ~t~ln B1 2, so that no dissociation of the ba~~ria~i~in Bi 2 complex can take place. In the reaction: binder + vitamin B1 z ~binder~vi~min B, 2 , dissociation of the complex formed can occur, especially with coated charcoal where the “free” compound is adsorbed 18). A distinct advantage of the assay compared with the bioassay is that the vitamin B, 2-binding property of the bacteria is unaffected by drugs, where as in the bioassay erroneous values are found when some drugs are present. Acknowledgements The authors wish to thank Mr C.J.M. van Gasteren, Philips-Duphar B.V., Weesp, The Netherlands for preparation of the dried Luctobuc~~~usZeic~~~~~i~, and Miss G. Helmus, Laboratory for Medical Microbiolo~, Groningen, The Netherl~ds for cul~vati~g Luetobuc~Z~uscasei. References 1 2 3 4 5 6 7 8 9
S. Kashket, J.T. Kaufman and W.S. Beck, Biochim. Biophys. Acta, 64 (1962) 447 M.S. McCall, J.D. White and E.P. Frenkel. Proc. Sot. EXP. Biol. Med., 134 (1970) 536 E.P. Frenkel, M.S. McCall and J.D. White, Am. J. Clin. Path& 53 (1970) 891 British Pharmacopeia, The Phsrmsceutieal Press, London, 1968, p. 264 M. Ceska and U. Lundkvist, Clin. Chim. Acta. 32 (1971) 339 E.P. Frenkel, S. Keller and MS. McCall. 3. Lab. Clin. Med., 68 (1966) 510 K. Kitabara and T. Sasaki, J. Gen. Appi. Microbial., 9 (1963) 213 Ram-Seng Lau, C. Gottlieb, L.R. Wasserman and V. Herbert, Blood, 26 (1965) 202 T. Sasaki. J. Bacterial., 109 (1972) 169
Clinica Chimica Acta, 55 (1974) 155-163 @ Elsevier Scientific ~blishing Company, Amsterdam - Printed in The Netherlands
CCA 6465
COMPETITIVE PROTEIN-BINDING ANALYSIS OF VITAMIN Bit USING ~CTO~ACILLUS L~ICHMA~~II AS A SPECIFIC BINDER
D.F.M. VAN DE WIELa, J.A. DE VRIESb, M.G. WOLDRINGa and I-LO. NIEWEGC aCentml Isotope Laboratory, State University, Groningen, bPhilips-Duphar B. V., Weesp and ‘Division of Hematology, Department of Medicine, State University, Groningen (The Netherlands~ (Received March 3,1974)
Summary Vitamin B1 2 was assayed by the competitive protein binding method, using Lactobacillus leichmannii as binding agent. The intact bacteria were used as “solid phase”, and separation of bound and free vitamin B1 2 was obtained simply by filtration of the bacteria. Filtration could be rapidly done using a disposable syringe provided with a filter. Optimal conditions with respect to the extraction of vitamin B1 2 from serum and binding of the vitamin to the bacteria were determined. The effect of several drugs and hormones on the assayed vitamin B1 2 concentration was investigated. The correlation with the Lactobacillus leichmannii bioassay was calculated.
Introduction The growth rate of Lactobacillus leichmannii is the parameter commonly used in the microbiological assay of vitamin B1 2 {the bioassay). This communication describes an assay for vitamin Bt 2 which utilizes the method of saturation analysis, where La~tobacilZus leichmannii is employed as a specific binder . . for vitamin B, *. It was found by Kashket et al. [l], that the uptake of [ 5 ’ Co] vitamin B1 2 by the microorganism depends on the amount of nonradioactive vitamin B1 2 which is present in the incubation medium. This property makes it possible to determine vitamin B I 2 by means of competitive protein binding analysis, using the intact bacterium as “solid phase”. Separation of free from bound vitamin B1 2 can be done either by filtration or by centrifugation. McCall et al. [23 described this kind of assay for serum folate, and gave it the name of radiomicrobiological assay.
156
Materials and Methods
vitaminB1 *
standard solutions A stock solution was prepared by dissolving crystalline cyanocobalamin (Merck, Darmstadt, p.a.) in distilled water after which the concentration was determined by spectrophotometry. The solution was further diluted to a vitamin B1 2 concentration of 2000 pg per ml. This stock solution was kept in the dark at 4”. Standard solutions of O-1000 pg vitamin B1 2 /ml were freshly prepared every month by diluting 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml stock solution with distilled water to 20 ml. [5 ’ Co] Vitamin B1 z was obtained from Philips-Duphar B.V., Petten, The Netherlands, with a specific activity ranging from 100 to 300 mCi/mg. Purity and specific activity were examined by the method of Frenkel et al. [3]. The radiochemical purity was examined by the method of the British Pharmacopeia [ 41 and proved in all cases to be more than 90%. Before use the [’ 7 Co] vitamin B1 2 solution was diluted with water until the activity was 0.2 I.tCi/ml. 0.35 M acetate buffers 47.628 g CH3 COONa * 3Hz 0 (Merck, p.a.) was dissolved in 2 litres of distilled water. 1 litre of the solution was brought to pH 4.7 and 1 litre to pH 5.1 with concentrated HCl. Before each experiment 1 ml 1.0% KCN solution was added to 100 ml of each buffer. Lactobacillus leichmannii (American Type Culture Collection 7830) 0.2 ml of a suspension of Lactobacillus leichmannii (absorbance 0.600) was inoculated into 10 ml medium, which was a sterilised solution of 10 g glucose, 10 g yeast extract (N.S.G.F.), 1 ml Tween 80, 0.25 g thiomalic acid and 0.1 mg thymidinephosphate in 1 litre of water (final pH 5.3). The bacteria were incubated for 20 h at 37” after which the whole mixture was transferred to 1 litre of medium. After three days incubation at 37” the bacteria were washed with saline, and dried for 4 days at 37”. For use in the vitamin B1 2‘ assay, 10 mg bacteria were suspended in 10 ml 0.07 M phosphate buffer pH 7.0. The bacteria were centrifuged and resuspended several times, until the supematant remained clear. 1 ml of the stock suspension was mixed with 9 ml phosphate buffer pH 7.0, giving a bacteria content of about 0.1 mg/ml (dry weight). The suspensions were kept at 4” and were stable for at least three months. Serum ex traction 2 ml serum was mixed with 4 ml 0.35 M acetate buffer pH 4.7 containing 0.01% KCN in a glass tube, which was stoppered with a perforated plastic cap. The tube was kept in a boiling water bath for 15 min and then centrifuged (2500 rev./min). 3 ml of the supematant was pipetted into a plastic tube. Incubation 1 ml standard vitamin B1 2 solution (O-1000 pg/ml) was mixed - in duplicate - with 2 ml 0.35 M acetate buffer pH 5.1 containing 0.01% KCN in a
IO-* Fig. 1. Plastic disposable syringe, used for incubation (1 and 2) and filtration tube with incubation mixture: B: plastic adaptor with filter.
ml.Lact.1. (3)
-
of bacteria.
A: plastic
Fig. 2. Saturation curve of Lactobacillus leichmannii. 0.01-0.1 ml bacteria suspension (0.1 mg/ml) incubated with 30 pg [5 7Co] vitamin B12. All points were obtained from duplicate determinations.
was
plastic tube. To all standards and extracts 0.02 ml [’ 7 Co] vitamin B1 2 dilution (about 15 pg) was added plus 0.02 ml bacteria suspension (0.02 ml suspension binds about 50% of the [’ 7 Co] vitamin B1 2’). After mixing with a Vortexmixer the content of each tube was taken up in a plastic disposable syringe, and incubated for 120 min at room temperature. Separation of bound from free vitamin B1 2 During the incubation the syringes were provided with specially prepared filters (Selectron filter GF92, diameter 10 mm, C. Schleicher and Schiill, Dassel, G.F.R.) (see Fig. 1). After the incubation the mixtures were forced through the filters, and the filters were counted in a well-type gamma scintillation counter. Results The standard curve For the preparation of a standard curve it was necessary to determine the amount of bacteria that gave an optimal binding under the experimental conditions. This was done by making a “saturation curve” (Fig. 2). There appeared to be a reasonably good linearity up to a binding of 50%, which was obtained with 0.02 ml suspension. For this reason 0.02 ml suspension was used for the standard curve (Fig. 3). In one experiment filtration was compared with centrifugation. In the latter case 2 ml supematant was measured in a counter (Table I). The percentages bound after centrifugation were nearly the same, but in general filtration gave better results than centrifugation. For this reason and also because of its convenience we preferred filtration as the method of separation.
158
01.1, 0
200
400
600
&z(pg)
800
fOO0
-
Fig. 3. Standard curve with ~ctobac~l~us Zejc~~annii. 0.03 ml bacteria suspension was incubated with 30 pg [57Co]vitemin B12 and O-1000 pg unlabelled vitamin BI 2.
~~~~~~tio~ time, pH and temperut~re The incubation time was determined with the aid of several standard curves (O-1000 pg vitamin B1 2 ) which were obtained after various incubation ,times (Fig. 4). After 120 min the slope of the curve remained nearly constant, therefore 120 min was chosen as the incubation time. The influence of the pH on the binding was determined with 0.07 M acetate buffers (pH 3.5-6.0) and 0.07 M phosphate buffers (pH 5.0-8.0) (Fig. 5). It appeared that, in acetate buffer, the binding was strongly pH-dependent. Variation of the binding with temperature was determined by making three standard curves, at lo”, 21” and 35” with a fixed incubation time of 120 min (Fig. 6). Incubation at room temperature gave the best standard curve. Ex
trffction The method of extraction should meet the following requirements: (a) The serum proteins must precipitate qu~titatively, because of the
TABLE 1 SEPARATION TRATION
OF FREE AND BOUND
VITAMIN
B12 EITHER BY CENTRIFUGATION
OR BY FIL-
Vitamin BIZ @g) 1000
0
100
200
400
W Bound (t S.D., n = 2) after filtration
‘to.1 f 1.3
28.7 f 1.6
22.7 zk0.7
15.3 f 0.4
8.7 f 0.4
% Bound (t S.D., n = 2) after centrifugation
41.9 f 0.3
30.6 f 0.7
24.5 f 0.7
16.7 f 0.3
10.4 * 0.4
159
80
240 min //790 I) -130 I‘ -60 II \ 30 I,
20
t t
01 OO_L Blz(pg)
’
,
L
f
3.5 4
5
6
7
5
8
PH -
-
Fig. 4. Variation of the standard curve with the time of incubation. Fig. 5. Percentage bound as function of PH. lA,
acetate buffer; X -X
, phosphate buffer.
interference of soluble proteins with the binding between bacteria and vitamin 3, 2 (see below). (b) After centrifugation the volume of the supematant must be as large as possible because the sensitivity of the assay is such that much vitamin B1 2 is required. (c) The efficiency of the extraction must be reasonably constant, to avoid individual correction for each serum. (d) The extraction buffer must allow a good binding between bacteria and vitamin B, *.
0
1000
500 812 (pg)
-
Fig. 6. Variation of the standard curve with the temperature 0-e 10”: X-X 36O. . 21°;~------+
at a fixed incubation time of 120 min.
160
Buffers Two buffers were examined, glutamate [5], and acetate buffer according to Frenkel no precipitation of the serum proteins which the centrifugation step. The soluble protein, with the binding between vitamin B 1 2 and glutamate buffer was abandoned.
buffer according to Ceska et al. et al. [6]. Glu~a~ buffer gives offers the advantage of omitting however, appeared to interfere bacteria, and for that reason the
The su~ernatant uo~ume as function of pH and molarity 0.7 M phosphate buffers with pH 4.5, 5.0, 5.5, 6.0 and 7.0 were mixed with serum and treated as described under Materials and Methods (phosphate buffer was chosen at first, because the bacteria showed better B1 2 binding properties in phosphate than in acetate buffer). In addition several acetate buffers (pH 5.5) with molarities of 0.7, 0.35, 0.0’7 and 0,007 were mixed with serum and treated in the same way (Fig. 7). The largest supernatant was obtained with pH 4.5 and 0.35 M. Influence of molarity on the standard curve Three standard curves were prepared in potassium phosphate buffers (pH 7.0) which were saturated or with molarities of 0.7 and 0.07 (Fig. 8). There was a large difference between saturated and 0.7 M, but molarities between 0.7 and 0.07 did not appear to be critical. Efficiency of the extraction The efficiency of the extraction was determined by preincubating 2 ml serum with 30 pg [” ’ Co] stein B 1 2 for 30 min, followed by extraction as described under Materials and Methods. The supernatant was counted for radioactivity and the efficiency of the extraction, i.e. the percentage [’ 7 Co] vitamin B1 2 in the supematant, was calculated.
70-
30-
20 0
$1
* (1 300 812(P9)
Fig. 7. Volume of the supematant as a function of PH end molarity of the buffer. Fig. 8. Variation of the standard curve with the buffer molarity.
11 600 -
$1 900
161
Repeated extraction of one serum gave an extraction efficiency of 88.8 + 1.1 (S.D.). Extraction of six different sera gave an efficiency of 89.4 rt 2.9 (S.D.). The efficiency of the extraction appeared to depend on the ratio volume serum:volume buffer. In one serum this ratio was varied as 1: 1,1:3 and 1:7, giving an extraction efficiency of respectively 93.2, 98.2 and 99.2%. Consequently 0.35 M acetate buffer pH 4.7 was chosen as the extraction buffer because: (a) the supernatant volume was sufficient large, (b) a good standard curve could be obtained with this buffer, and (c) the extraction efficiency was reasonably high and constant.
The precision of the standard curve in the region of O-100 pgjml was found to be + 10 pg (SD.). The 95% confidence limit is therefore 20 pgjml, so that values below 20 pg/ml must be considered indistinguishable from zero (see also Discussion). Accuracy Pooled serum was diluted with distilled water to 80, 60,40 and 20% after which the vitamin B1 2 content was determined. In undiluted serum the vitamin Bi 2 content was 680 pg/ml. In the diluted sera the values found were respectively 540, 420, 300 and 220 pg/ml where as the calculated values were 540, 408, 300 and 136 pgfml. The accuracy was also tested by comparing the results with that of the “bioassay”. In the bioassay use is made of the fact that the growth of Lac~obac~f~us Ze~c~~a~~~~depends on the amount of vitamin B1 2 in the growth medium. Although in both assays, competitive protein-binding as well as bioassay, the same bacteria were used, the correlation between them was poor (Fig. 9). Precision A pool was made of 30 different sera, after which the vitamin B1 2 con-
800
x
t
i 600
(Ph
.
Z” a m,
0
(Y
400 i 200
i 0-b
0
. .
.
‘ii ii
l
l
t
l
.
l
..
. Q
.* b’
.
5
.
I
200 Radio
I
I
400
I
I
I
600
Bvz (Pg) m~crobioiog~cal
I
800
I
I
1000 assay
Fig. 9. Correietionbetween the competitive protein-biidiig assay and the bioassay. The equation of the regressionline wes y = 0.686x - 8.4. The correlationcoefficient was 0.756.
162 TABLE
II
PRECISION During
OF
the
are values
THE
seven
RADIOMICROBIOLOGICAL
days
obtained
between
from
duplicate
assay
ASSAY
I and
II the
sera
were
stored
at -2O’C.
Figures
Serum
Assay
N.
1000
I
Assay
665
(645.
685)
730
de G.
395
(390.
400)
380
Ha.
403
(375.
430)
330
O(
He.
brackets
II
970
Sch.
U.
between
determinations.
355
0. (355,
0)
20
355)
(
0.
40)
415
de V.
985
(970.1000)
R.
350
(300,
400)
350
D.
630
(575,
685)
675
P.
308
(305,
310)
340
A.
418
(390,
455)
430
K.
540
Sm.
395
(380,
410)
310
G.
590
(565,
615)
615
1000 (350.350)
515 (560,670)
tent was determined in twelve-fold: 379 f 45 pg/ml (S.D.). The vitamin B1 2 content of a series of sera was determined in duplicate. After storage of the sera at -20” during one week, the determination was repeated (Table II). Influence of drugs and hormones Several drugs and hormones were added to different aliquots of 2 ml serum, after which the vitamin B1 2 content was determined. The weight of the drugs was chosen such that it corresponded with a mean serum content after
TABLE
III
INFLUENCE
OF
DRUGS
AND
HORMONES
ON
THE
VITAMIN
B12
RADIOMICROBIOLOGICAL
ASSAY Added
per ml serum
Serum
0.0
495
0.4
mg tetracycline
HCI
0.1
mg barbital-
0.3
mg tolbutamide
525
1.0
mg sulfamerazine
490
Serum 420
500 520
4.0
~g nitrazepam
0.5
mg promazine
2.0
/lg acenocumarolum
535
2.0
mg salicylic
455
25.0
1 (pg B I z /ml)
525 HCl acid
ng oestradiol
515
440
6.0
ng testosterone
425
3.0
ng digoxine
412
1.0
/Ig progesterone
470
ng folic
485
30.0 0.1 10.0 1.4 107.0
acid
pg cortisol
417
l.(g diazepam
415
ng triiodothyronine
422
ng thyroxine
440
2 (pg BIZ
/ml)
163
maximal dosage, while the weight of the added hormones corresponded with the upper limit of the normal serum content (Table III). The values that were found all appeared to be within the range of precision. Discussion The sensitivity of the assay was not very high, therefore it was necessary to extract at least 2 ml serum. Several methods were tested to increase the sensitivity. First the manner of cultivat~g the bacteria was changed. ~~~~0~~~~~~~s ~e~c~~u~~~~is usually c~tiva~d in a vitamin 3, 2 ~ont~ning medium 191. To prevent a possible satumtion of the bacteria with vitamin B1 ?, thymidine phosphate [l] was substitu~d for vitamin B1 2. No large difference was found between “thymidine” and “Bl 2 ” grown bacteria: the decrease of the percentage bound between 0 and 1000 pg vitamin B1 2 was respectively 31.8 and 37.5. In addition another vitamin Bi 2-binding bacterium was tested, which is known to be able to grow on a vitamin B 1 z’-deficient medium, namely Lactobacillus casei [71. The sensitivity of Lactobacillus casei however was much less: the decrease of the percentage bound was 19.0. An advantage of the assay is the very rapid separation of free and bound . . ~t~ln B1 2, so that no dissociation of the ba~~ria~i~in Bi 2 complex can take place. In the reaction: binder + vitamin B1 z ~binder~vi~min B, 2 , dissociation of the complex formed can occur, especially with coated charcoal where the “free” compound is adsorbed 18). A distinct advantage of the assay compared with the bioassay is that the vitamin B, 2-binding property of the bacteria is unaffected by drugs, where as in the bioassay erroneous values are found when some drugs are present. Acknowledgements The authors wish to thank Mr C.J.M. van Gasteren, Philips-Duphar B.V., Weesp, The Netherlands for preparation of the dried Luctobuc~~~usZeic~~~~~i~, and Miss G. Helmus, Laboratory for Medical Microbiolo~, Groningen, The Netherl~ds for cul~vati~g Luetobuc~Z~uscasei. References 1 2 3 4 5 6 7 8 9
S. Kashket, J.T. Kaufman and W.S. Beck, Biochim. Biophys. Acta, 64 (1962) 447 M.S. McCall, J.D. White and E.P. Frenkel. Proc. Sot. EXP. Biol. Med., 134 (1970) 536 E.P. Frenkel, M.S. McCall and J.D. White, Am. J. Clin. Path& 53 (1970) 891 British Pharmacopeia, The Phsrmsceutieal Press, London, 1968, p. 264 M. Ceska and U. Lundkvist, Clin. Chim. Acta. 32 (1971) 339 E.P. Frenkel, S. Keller and MS. McCall. 3. Lab. Clin. Med., 68 (1966) 510 K. Kitabara and T. Sasaki, J. Gen. Appi. Microbial., 9 (1963) 213 Ram-Seng Lau, C. Gottlieb, L.R. Wasserman and V. Herbert, Blood, 26 (1965) 202 T. Sasaki. J. Bacterial., 109 (1972) 169