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Vitamin B12 determination by means of radioisotope dilution and ultrafiltration
CLINICA
CHIMICA
1’ITAMIN AND
171
ACTA
B12 DETERMINATION
BY MEANS
OF RADIOISOTOPE
DILUTION
ILTRAI~ILTRATION
The vitamin B,, concentration in serum is determined by a new procedure, The vitamin is using the competition for intrinsic factor with [S7Colcobalannn. liberated from its binding to serum protein by heating at pH 4.9 in the presence of cyanide. [5’Co_B,, and intrinsic factor are added, and the solution is ultrafiltered tin-ough a dialysis membrane. The amount of B,, in the sample can be calculated from the radioactivity of the ultrafiltrate compared to a standard curve. The optimal conditions
and the methodological
errors of each step in the procedure
are examined.
In view of the technical difficulties encountered in microbiological determination of vritamin B,, in serum, several attempts have been made to replace this method by methods of determinations more suited for routine clinical use. These methods are based on the competition between unlabelled and radioactive I~,, for the linkage to specific l&,-binding proteins. However, some of these methods are too complicated and time-consuming for routine use, whereas others do not give reliable results. For these reasons, the following method was devised. The new method is comparatively simple to perform, and the results of the determinations are in good agreement with those found by microbiological determination. METHOI)
Reagera fs r. Acctatc ha@&, $H 4.8,0.3 -11, to which are added 20 /'g of KCN per ml solution. 2 Sodiwn chloride 0.9”; (saline). 3. B,, stmdavd solzttiom: The concentration of the commercial solution is determined calorimetrically. The solution is diluted with water to contain, o, 50, IOO, 200, 300, 500, 700, I000 pg of B,, per ml. Stable several months at +5”. 4. (n) I 5’Co~B,,, stock solution: IO i’C of :~ XojB,, (Code C T 2 P, specific activity about qo ,LC//J~, from The Radiochemical Centre, Amersham, England) are diluted with water to contain IOOO pg/ml. The solution is divided into small portions (I-Z ml) and stored at -20".
172
I’RIIJI)KKR
d
d.
~~~2~~~0~2~ Stock solution (R) is thawed and diluted wit11 (b) ~~~CO]B,,,7~~0~~~~~ water to contain about 350 pg/ml. Stable for at least one week in the refrigerator. 5. Intrinsic Factor IF /. (We use IF from AS GEA :s, Copenhagen, Denmark.) (n) Stock solution: IOO q of Iii in IOO ml of o.c,‘){~ NaCl (2). The solution is centrifuged until the supernatant is perfectly clear. This is divided into I.S-ml portions in test tubes and stored at -zoo, when it is stable for at least six months. This solution must be used i~lllllediat~~l~ after thawing and cannot be frozen twiw. (h) ll~o&itzg soZz&o~: 1 ml oi stock solution is first diluted with o.t~“;, NaCl (2) to IOO rd. This solution is further diluted with saline to contain tlw amount of IF determined by titration, as described below. These solutions must be prepared inrmediately before use and kept in an ice-bath.
The adequate quantity of IF (sb) is determined in the following way: ‘l%t primary working solution of IF (jb) is diluted to contain I, 2, 3, 4, 5, 6, 7, S and I0 c!g of II; per ml o.9”0 NaCl solution. I.5 ml of acetate buffer with cvanide (I), 0.5 ml of water, 0.j ml of saline and 0.5 ml of !!.~Xo i R,, working solution (lb) are pipetted into IO test tubes. To the first of the tubes is added 0.5 ml of 0.9 O,i SaCl solution and to the others 0.5 ml of the nine IF dilutions. After mixing, the tubes are left at room tempera.ture for IO min. Dialysis tubing is moistened to softness in water, cut into pieces of about ag cm length and carefully wiped off. One end of each piece is closed by firmly knotting the tubing. Three 1~~1 of the sample are introduced through the open end, wliereupon tllis countsjmin 5500
1
VITAMIN
I73
R,, DETERMINATION
end is also closed by a firm knot. A similar method has been described The tubing is folded double, and introduced
into a centrifuge
previously1>2.
tube in such a way that
the knots remain outside the brim. It is fixed in this position by a rubber stopper. During centrifugation, the tubing should be hanging in the tube. The tubes are centrifuged in a refrigerated centrifuge with swing-out head for 75 min at IOOO g at a temperature not exceeding 15'. Generally, the volume of the ultrafiltrate amounts to about I ml. 0.5 ml of the ultrafiltrate is transferred to test tubes and made up with water to a volume suitable for the well counter. The activity is determined in a well type gamma scintillation counter. The results are plotted, and a curve is drawn as exemplified in Fig. I. The dilution of IF containing the amount able to bind about 80’$;; of the B,, is chosen for the determinations (as compared to tube I without IF). For the example given in Fig. I, this quantity is 4.5 /“g/ml. Before starting the determinations, this quantity must be controlled and corrected, if necessary, as described below. This “titration” of IF is made for every new batch of j%olB,, and for every new batcll
of IF.
Prejaratiofz
of stadard cawve 0.5 ml of o.go;b NaCl solution (z), 0.5 ml of each B,, standard solution 0.5 ml of L%~]B12 working solution 1.5 ml of acetate buffer with cyanide After mixing, 0.5 ml of the adequate tion is added. The samples are left at room the radioactivity of the filtrate is measured A standard curve is plotted, and drawn as
(3) o-1000 pg/ml; (4b), and (I) are pipetted into test tubes. working IF dilution (5b) found by titratemperature for IO min, ultrafiltered, and as described above for “titration” of IF. exemplified in Fig. 2. This standard curve
ccunts/min 30002500.
.A ,/
2000.
./
1000. 1500.
/
500. ./
0’.
0
l:icb, 7 /, -.
.
loo
. 200 300 Loo sol 600
700
m
900
.
lcal
P9 42/m' Standard curve for BLn solutioIls trerttetl sccortliIlg to the proposer1 method.
is prepared with every series of determinations. Provided that the optimal amounts of IF are chosen, different quantities of the same serum should give identical concentration values. Scmnt samples I ml of water, I ml of serum and 3 ml of acetate buffer with cyanide (I) are mixed in a centrifuge tube and heated in boiling water for 20 min. ,4fter cooling to
room temperature, the tubes are centrifuged, and 2.5 ml of the clear supernatant ar(’ transferred to a test tube. 0.5 ml of Ij’CojH,, working solution (412) is added. ,lftcr mixing, 0.5 ml uf the chosen working IF dilution (5 13)is added, follo\ved t)h, auothc~I mixing. After 10 min, the samples are ultrafiltered and measured as descrilwd ah)\~, for “titration” of 11’. The I_‘,,, concentration is read from the standard cu~-\.c. counts/min 16CO-
600. 600. LOO. 200 i
OJl-
L.25
COSTROL
CM
L.75
I
5MI
5.25
.
5.50
5.75
.
6.00
.
6.25
<
691 pH
I
In order to obtain optimal liberation of vitamin B,, from its bincling to serui11 proteins, we studied the influence of pH on the release of tile vitamin. To I ml of a normal ljuman serum mere added I ml of water and 3 ml of 0.2 :lf acetate buffer solutions of pH 3.8~5.0. The pH of the nlistures was tleterminc~d electrometricall~~. The samples were heated and ccntrifugcd as dcscrilwd ahove, and the pH of the decanted supernatants teas corrected to pH ;.I 11~ ~arelul addition of T S NaOH or HCl. 57Co ‘I$, and 11; were added, and tllrb anal!-sis completed a> described above (Fig. 3). Optimal liberation of \+aniin I_‘,,,was obtained in the tub+
VITAMIN
R,,
DETERMINATION
I75
heated at pH 4.75 to 5.00, corresponding to pH 4.5 and 4.8, respectively, in the added acetate buffer solutions. The experiment recorded in Table I demonstrates that no B,,
is co-precipitated with the protein. For the binding of B,, to IF the acidity of the solution is of little importance. A special series of standard solutions containing 300 pg H,, per ml was analysed, using a constant amount of IF and 157CoyB,,, but with a pH varying from 4.75 to 5.3. The results differed only within the variations of the method.
Standard
curves were prepared
as described
above,
excluding
cyanide.
These
curves were identical with those including cyanide. Heating the standards for 20 min, as described for the serum samples, did not affect the standard curve, neither in the absence of cyanide nor in its presence. Consequently, neither boiling nor the presence of cyanide is of any influence on the vitamin B,, or on its binding to IF. However, confirming previous investigators (e.g. “) we found that the presence of cyanide is necessarv for a complete release of B,, from the serum proteins. Serum samples were run in parallel, on one hand according to the standard procedure, and on the other hand omitting the cyanide in the buffer. \Vithout cyanide, the results were constant11 lower, ranging from 909; down to 25”; of the lralues obtained with cyanide added to the buffer-. (3) Ionic strcngtlz The ionic strength of the solution is of some importance for the binding of B,, to II’. To a series of standard solutions containing 300 pg of B,, were added the same amounts of IF and I~‘CO]B~~ as for the serum samples, and the same volume of a pH 4.8 acetate buffer solution with a concentration varying from 0.1 to 0.5 mole per liter. The solutions
were ultrafiltered as described above, and the filtrates were counted. The difference did not exceed 15Ti, with a minimum count (highest binding) with buffer solutions containing between 0.30 and 0.35 mole per liter, corresponding to a molarity of the final solution of 0.170 to O.I()j mole per liter. Consequently, we have chosen a final molarity of 0.170, which is also sufficient to maintain a stable pH in the serum samplei, equalling that in tile standard solutions. (4) L:ltmJiltvutiou In order to check the ultrafiltration procedure, z ml of [S7Co]B,, working solution were added to test tubes containing either z ml of 0.9~~ KaCl solution or 2 ml of 5 differrnt sera with varying protein concentrations. 6 ml of acetate buffer were added to each tube. The samples were lleated for 20 min in boiling water, and centrifuged after cooling. The radioactivity of 0.5 ml of the supernatants was measured, and 3 ml of the supernatants were ultrafiltered, as described above. 0.5 ml of the ultrafiltrates and 0.5 ml of the contents of the bags were measured as above. The results arc recorded in Table I. They demonstrate that the counts recorded in the ultrafiltrates are only 77 to So”, of the expected ones, whereas the activity of the remaining content inside the bags is fairly equal to the supernatant after centrifugation. Rx- continued centrifugation at an increased speed we found that the reason for this;‘differexe is due to dilution of the ultrafiltrate with moisture in the watersoaked dialysis tubing. Hence, it is of importance to keep centrifugation speed and time reasonably constant.
176
The reproducibilit\of thr method was clrecked by dcterniination on two different days of a scrics of 3j samples Lvithin tlrt, normal range. Thc,rc u’as no signiticant difference between tllc mean values. The standard deviation was rc) pgjml, giving a coefficient of variation 0I +o”,,. Sera were analT-zed both according to the standard abovc~, using r ml of Yiamplr, and also using 0.5 1111 or z ml
of the differencxlb
procedure, of
sllllplr.
as tlcscril~ctl -1-o
tire
tlll,c?
with I 1111of sample, I ml of n-atcr mxs added, to tlrt, 0.5-nil tubes 0.5 1111of ?;a(‘1 and I ml of water were added, and in the 2-ml tub the water \vas olllittetl. A4ll tulx5 were analvsed according to the standard lmmdure. _4 con~p-ison lxhxen tllcl r~~sults is demonstrated in Fig. 4. Results of determinations of sera witll added unlal~rllctl I?,, arc’ report~tl in Table II. The mean recover!’ was ()_c(),,. w B&ml 800-
Euglena gradsmethod ~gQz/m~ 700. . 600. / soo-
J*' .
.
. .
.'* . .
VITAMIN
B,,
UETERMISATIOIi
‘77
Serum samples were analysed both according to the present method and to a microbiological method using Eugle~za gvncilis*. The results are recorded in Fig. 5. In serum samples from 68 apparently healthy persons the mean value was 473 pg/ml, SE + 16 pg/ml, SD + 135 pgjml, range 160~665 pg/ml.
A method for determining vitamin K,, in serum using a radioisotope dilution technique requires the following steps: I. Liberation of the vitamin from its protein binding; II. Addition of a fixed amount of labelled vitamin I~,,; III. Addition of a B,,-binding protein; IV. Separation of free from bound B,,; T’. Calculation of the original vitamin concentration from a measured radioactivity. I. According to the results described under CONTROL ENPERIMESTS, optimal libevatiorz of vitamin R,, was achieved by heating at a pH around 4.9 (buffer solution pH 4.8). Table I demonstrates that no vitamin B,, is co-precipitated with the protein at this pH. This pH is also suitable for the binding to and the stability of IF. At this pH, vitamin B,, is stable*. Without heating, the liberation was far from complete. The presence of cl-anide was found essential for total liberation of the vitamin. It is also to be expected that transformation to the cyanide form will contribute to equalization of the binding tendency and capacity of different cobalamine derivatives occurring in serum. We also found that the method introduced by Rothenberg” and modified by Frenkel et al.5-including boiling of the sera at two different l)H-apart from being more time-consuming, sometimes failed to give clear supernatants, and did not increase the recovery. A modified H,, liberation procedure, using acetate buffer pH 4.1, proposed
178
PRIEDNER
Ct Cd.
by KarstofP, is likely to give lower values, since it does not include the presence of cyanide. This is confirmed by the comparatively low normal range reported. \I:c also found the method of Lau rt al.’ to give erroneous results, probably because of the low pH used for liberation of the vitamin from serum protein and the omission of cyanide. II. The best precision and vc~jwoducibilifv of the metliod was achieved Lvlien the added amount of labelled I& was appro&ately equal to the mean (quantit! of IS,, present in normal samples. A larger addition of labelled I&, decreases the sensitivity of the method, nhereas a smaller quantity also decreases tlie reliability b~,r lowering the radioactivity to be measured. The quality of the labelled vitamin K 12 is of great importance for the resu1t.s. Fig. 1 illustrates curves of titration of 11; with two different commercial preparations of cobalamin. With the preparation used by us, a nearly complete absorption was achieved, \\hereas 1 I”,, of the radioactivity remained fret, when the other prclxration was used, even with a large excess of I I;. III. For the s/mijic hiudiu, 17of R,, xve have chosen intrinsic factor, since tlris protein is commercially available, and since it gives a stable and stoichiomctri(~ coupling with the vitamin. Sormal serum, used by Frenkel et nd.j, as well as myeloic serum”, are binders containing several proteins with affinity to the vitamin. IF is unstable in diluted solutionH, but this difficult\- is avoided by storing the stock solution in the frozen state, and making up a working solution for each series of determinations. The quality of tile IF is of paramount importance. The presence of other proteins may be of unpredictable influence on the binding stabilit!, and capacity, and different commercial preparations have different stability. The oncwe now use lias given repro&cible results wlien strict]\, adhering to the standard proccdurc. This is btrcau~ me carefullv centrifuge the stock solution and discard an\. undissolved residue, ii procedure ethic11 seems to have been neglected by previoux authors. \I’hen and if a crystalline preparation of IF will be available, this will lx-ol~al~l~~be preferable. IV. 1;or S?/!W&~lZ Of‘fJWfYfY & from that bound to intrinsic factor, our motlification of the 12chlm-g1 mctllod as modified by Sterling c? nl.” proved to 1~ con\:enicwt, and to gi\e I-eproducible results. Tllc loss of m”,, of tlrc activit\. b17 I nltraiiltraticm througlr tlw dialysis tubing ih csplained 13!- tlw fact that tllci tubing ahorbs sonitwater wlicii it is iiioistcwd. This water dilutes the ultrafiltrate lx m”,, of its voluine, since it constitutes about 200 mg of the I ml \-olume of filtrate. ‘171~~ reproducibilit~~ of tlie radioactivity of the filtrate from standard solutions, as \vell as tlrat from struni samples, dcmonstratcs that tlie degree of dilution is constant, and that coiilpariso~l of sample values with standard values gives correct results. In agreciiicnt \vitli 1iot1icn1~er~*, we obtainctl unrc~liable results xvhrn uGiig absorption of the free I:,, to active charcoal, as used I)>- I,au r? lr1.’ and by otlrcrs!‘~“‘. I-. In tlie prcsent iiictliod, the i~itauzill coucc~7ztvatio,z in tire sample c-allnot mail!, be calculated according to the formula of Rittenbcrg it nl. II because of tire dilution of the ultrafiltratc by water from the dialysis tubing. Howcvcr, comparison of tlrc activitlr with a standard curve, as described in the nictliod, was found convenient. Tlie present method is easy to perform, but recluircs facilities for rcfrigeratctl centrifuging and for detrrmitiation of gam~na radiation. Tt is \vcll suitctl fcor dctc>rmination of vitamin IS,, in series of I0 to 40 samples. .\ tecllnician can easilv anal\-sc 50 samples a dav.
VITAMIN
B,,
DETERMINATION
CHIMICA
1’ITAMIN AND
171
ACTA
B12 DETERMINATION
BY MEANS
OF RADIOISOTOPE
DILUTION
ILTRAI~ILTRATION
The vitamin B,, concentration in serum is determined by a new procedure, The vitamin is using the competition for intrinsic factor with [S7Colcobalannn. liberated from its binding to serum protein by heating at pH 4.9 in the presence of cyanide. [5’Co_B,, and intrinsic factor are added, and the solution is ultrafiltered tin-ough a dialysis membrane. The amount of B,, in the sample can be calculated from the radioactivity of the ultrafiltrate compared to a standard curve. The optimal conditions
and the methodological
errors of each step in the procedure
are examined.
In view of the technical difficulties encountered in microbiological determination of vritamin B,, in serum, several attempts have been made to replace this method by methods of determinations more suited for routine clinical use. These methods are based on the competition between unlabelled and radioactive I~,, for the linkage to specific l&,-binding proteins. However, some of these methods are too complicated and time-consuming for routine use, whereas others do not give reliable results. For these reasons, the following method was devised. The new method is comparatively simple to perform, and the results of the determinations are in good agreement with those found by microbiological determination. METHOI)
Reagera fs r. Acctatc ha@&, $H 4.8,0.3 -11, to which are added 20 /'g of KCN per ml solution. 2 Sodiwn chloride 0.9”; (saline). 3. B,, stmdavd solzttiom: The concentration of the commercial solution is determined calorimetrically. The solution is diluted with water to contain, o, 50, IOO, 200, 300, 500, 700, I000 pg of B,, per ml. Stable several months at +5”. 4. (n) I 5’Co~B,,, stock solution: IO i’C of :~ XojB,, (Code C T 2 P, specific activity about qo ,LC//J~, from The Radiochemical Centre, Amersham, England) are diluted with water to contain IOOO pg/ml. The solution is divided into small portions (I-Z ml) and stored at -20".
172
I’RIIJI)KKR
d
d.
~~~2~~~0~2~ Stock solution (R) is thawed and diluted wit11 (b) ~~~CO]B,,,7~~0~~~~~ water to contain about 350 pg/ml. Stable for at least one week in the refrigerator. 5. Intrinsic Factor IF /. (We use IF from AS GEA :s, Copenhagen, Denmark.) (n) Stock solution: IOO q of Iii in IOO ml of o.c,‘){~ NaCl (2). The solution is centrifuged until the supernatant is perfectly clear. This is divided into I.S-ml portions in test tubes and stored at -zoo, when it is stable for at least six months. This solution must be used i~lllllediat~~l~ after thawing and cannot be frozen twiw. (h) ll~o&itzg soZz&o~: 1 ml oi stock solution is first diluted with o.t~“;, NaCl (2) to IOO rd. This solution is further diluted with saline to contain tlw amount of IF determined by titration, as described below. These solutions must be prepared inrmediately before use and kept in an ice-bath.
The adequate quantity of IF (sb) is determined in the following way: ‘l%t primary working solution of IF (jb) is diluted to contain I, 2, 3, 4, 5, 6, 7, S and I0 c!g of II; per ml o.9”0 NaCl solution. I.5 ml of acetate buffer with cvanide (I), 0.5 ml of water, 0.j ml of saline and 0.5 ml of !!.~Xo i R,, working solution (lb) are pipetted into IO test tubes. To the first of the tubes is added 0.5 ml of 0.9 O,i SaCl solution and to the others 0.5 ml of the nine IF dilutions. After mixing, the tubes are left at room tempera.ture for IO min. Dialysis tubing is moistened to softness in water, cut into pieces of about ag cm length and carefully wiped off. One end of each piece is closed by firmly knotting the tubing. Three 1~~1 of the sample are introduced through the open end, wliereupon tllis countsjmin 5500
1
VITAMIN
I73
R,, DETERMINATION
end is also closed by a firm knot. A similar method has been described The tubing is folded double, and introduced
into a centrifuge
previously1>2.
tube in such a way that
the knots remain outside the brim. It is fixed in this position by a rubber stopper. During centrifugation, the tubing should be hanging in the tube. The tubes are centrifuged in a refrigerated centrifuge with swing-out head for 75 min at IOOO g at a temperature not exceeding 15'. Generally, the volume of the ultrafiltrate amounts to about I ml. 0.5 ml of the ultrafiltrate is transferred to test tubes and made up with water to a volume suitable for the well counter. The activity is determined in a well type gamma scintillation counter. The results are plotted, and a curve is drawn as exemplified in Fig. I. The dilution of IF containing the amount able to bind about 80’$;; of the B,, is chosen for the determinations (as compared to tube I without IF). For the example given in Fig. I, this quantity is 4.5 /“g/ml. Before starting the determinations, this quantity must be controlled and corrected, if necessary, as described below. This “titration” of IF is made for every new batch of j%olB,, and for every new batcll
of IF.
Prejaratiofz
of stadard cawve 0.5 ml of o.go;b NaCl solution (z), 0.5 ml of each B,, standard solution 0.5 ml of L%~]B12 working solution 1.5 ml of acetate buffer with cyanide After mixing, 0.5 ml of the adequate tion is added. The samples are left at room the radioactivity of the filtrate is measured A standard curve is plotted, and drawn as
(3) o-1000 pg/ml; (4b), and (I) are pipetted into test tubes. working IF dilution (5b) found by titratemperature for IO min, ultrafiltered, and as described above for “titration” of IF. exemplified in Fig. 2. This standard curve
ccunts/min 30002500.
.A ,/
2000.
./
1000. 1500.
/
500. ./
0’.
0
l:icb, 7 /, -.
.
loo
. 200 300 Loo sol 600
700
m
900
.
lcal
P9 42/m' Standard curve for BLn solutioIls trerttetl sccortliIlg to the proposer1 method.
is prepared with every series of determinations. Provided that the optimal amounts of IF are chosen, different quantities of the same serum should give identical concentration values. Scmnt samples I ml of water, I ml of serum and 3 ml of acetate buffer with cyanide (I) are mixed in a centrifuge tube and heated in boiling water for 20 min. ,4fter cooling to
room temperature, the tubes are centrifuged, and 2.5 ml of the clear supernatant ar(’ transferred to a test tube. 0.5 ml of Ij’CojH,, working solution (412) is added. ,lftcr mixing, 0.5 ml uf the chosen working IF dilution (5 13)is added, follo\ved t)h, auothc~I mixing. After 10 min, the samples are ultrafiltered and measured as descrilwd ah)\~, for “titration” of 11’. The I_‘,,, concentration is read from the standard cu~-\.c. counts/min 16CO-
600. 600. LOO. 200 i
OJl-
L.25
COSTROL
CM
L.75
I
5MI
5.25
.
5.50
5.75
.
6.00
.
6.25
<
691 pH
I
In order to obtain optimal liberation of vitamin B,, from its bincling to serui11 proteins, we studied the influence of pH on the release of tile vitamin. To I ml of a normal ljuman serum mere added I ml of water and 3 ml of 0.2 :lf acetate buffer solutions of pH 3.8~5.0. The pH of the nlistures was tleterminc~d electrometricall~~. The samples were heated and ccntrifugcd as dcscrilwd ahove, and the pH of the decanted supernatants teas corrected to pH ;.I 11~ ~arelul addition of T S NaOH or HCl. 57Co ‘I$, and 11; were added, and tllrb anal!-sis completed a> described above (Fig. 3). Optimal liberation of \+aniin I_‘,,,was obtained in the tub+
VITAMIN
R,,
DETERMINATION
I75
heated at pH 4.75 to 5.00, corresponding to pH 4.5 and 4.8, respectively, in the added acetate buffer solutions. The experiment recorded in Table I demonstrates that no B,,
is co-precipitated with the protein. For the binding of B,, to IF the acidity of the solution is of little importance. A special series of standard solutions containing 300 pg H,, per ml was analysed, using a constant amount of IF and 157CoyB,,, but with a pH varying from 4.75 to 5.3. The results differed only within the variations of the method.
Standard
curves were prepared
as described
above,
excluding
cyanide.
These
curves were identical with those including cyanide. Heating the standards for 20 min, as described for the serum samples, did not affect the standard curve, neither in the absence of cyanide nor in its presence. Consequently, neither boiling nor the presence of cyanide is of any influence on the vitamin B,, or on its binding to IF. However, confirming previous investigators (e.g. “) we found that the presence of cyanide is necessarv for a complete release of B,, from the serum proteins. Serum samples were run in parallel, on one hand according to the standard procedure, and on the other hand omitting the cyanide in the buffer. \Vithout cyanide, the results were constant11 lower, ranging from 909; down to 25”; of the lralues obtained with cyanide added to the buffer-. (3) Ionic strcngtlz The ionic strength of the solution is of some importance for the binding of B,, to II’. To a series of standard solutions containing 300 pg of B,, were added the same amounts of IF and I~‘CO]B~~ as for the serum samples, and the same volume of a pH 4.8 acetate buffer solution with a concentration varying from 0.1 to 0.5 mole per liter. The solutions
were ultrafiltered as described above, and the filtrates were counted. The difference did not exceed 15Ti, with a minimum count (highest binding) with buffer solutions containing between 0.30 and 0.35 mole per liter, corresponding to a molarity of the final solution of 0.170 to O.I()j mole per liter. Consequently, we have chosen a final molarity of 0.170, which is also sufficient to maintain a stable pH in the serum samplei, equalling that in tile standard solutions. (4) L:ltmJiltvutiou In order to check the ultrafiltration procedure, z ml of [S7Co]B,, working solution were added to test tubes containing either z ml of 0.9~~ KaCl solution or 2 ml of 5 differrnt sera with varying protein concentrations. 6 ml of acetate buffer were added to each tube. The samples were lleated for 20 min in boiling water, and centrifuged after cooling. The radioactivity of 0.5 ml of the supernatants was measured, and 3 ml of the supernatants were ultrafiltered, as described above. 0.5 ml of the ultrafiltrates and 0.5 ml of the contents of the bags were measured as above. The results arc recorded in Table I. They demonstrate that the counts recorded in the ultrafiltrates are only 77 to So”, of the expected ones, whereas the activity of the remaining content inside the bags is fairly equal to the supernatant after centrifugation. Rx- continued centrifugation at an increased speed we found that the reason for this;‘differexe is due to dilution of the ultrafiltrate with moisture in the watersoaked dialysis tubing. Hence, it is of importance to keep centrifugation speed and time reasonably constant.
176
The reproducibilit\of thr method was clrecked by dcterniination on two different days of a scrics of 3j samples Lvithin tlrt, normal range. Thc,rc u’as no signiticant difference between tllc mean values. The standard deviation was rc) pgjml, giving a coefficient of variation 0I +o”,,. Sera were analT-zed both according to the standard abovc~, using r ml of Yiamplr, and also using 0.5 1111 or z ml
of the differencxlb
procedure, of
sllllplr.
as tlcscril~ctl -1-o
tire
tlll,c?
with I 1111of sample, I ml of n-atcr mxs added, to tlrt, 0.5-nil tubes 0.5 1111of ?;a(‘1 and I ml of water were added, and in the 2-ml tub the water \vas olllittetl. A4ll tulx5 were analvsed according to the standard lmmdure. _4 con~p-ison lxhxen tllcl r~~sults is demonstrated in Fig. 4. Results of determinations of sera witll added unlal~rllctl I?,, arc’ report~tl in Table II. The mean recover!’ was ()_c(),,. w B&ml 800-
Euglena gradsmethod ~gQz/m~ 700. . 600. / soo-
J*' .
.
. .
.'* . .
VITAMIN
B,,
UETERMISATIOIi
‘77
Serum samples were analysed both according to the present method and to a microbiological method using Eugle~za gvncilis*. The results are recorded in Fig. 5. In serum samples from 68 apparently healthy persons the mean value was 473 pg/ml, SE + 16 pg/ml, SD + 135 pgjml, range 160~665 pg/ml.
A method for determining vitamin K,, in serum using a radioisotope dilution technique requires the following steps: I. Liberation of the vitamin from its protein binding; II. Addition of a fixed amount of labelled vitamin I~,,; III. Addition of a B,,-binding protein; IV. Separation of free from bound B,,; T’. Calculation of the original vitamin concentration from a measured radioactivity. I. According to the results described under CONTROL ENPERIMESTS, optimal libevatiorz of vitamin R,, was achieved by heating at a pH around 4.9 (buffer solution pH 4.8). Table I demonstrates that no vitamin B,, is co-precipitated with the protein at this pH. This pH is also suitable for the binding to and the stability of IF. At this pH, vitamin B,, is stable*. Without heating, the liberation was far from complete. The presence of cl-anide was found essential for total liberation of the vitamin. It is also to be expected that transformation to the cyanide form will contribute to equalization of the binding tendency and capacity of different cobalamine derivatives occurring in serum. We also found that the method introduced by Rothenberg” and modified by Frenkel et al.5-including boiling of the sera at two different l)H-apart from being more time-consuming, sometimes failed to give clear supernatants, and did not increase the recovery. A modified H,, liberation procedure, using acetate buffer pH 4.1, proposed
178
PRIEDNER
Ct Cd.
by KarstofP, is likely to give lower values, since it does not include the presence of cyanide. This is confirmed by the comparatively low normal range reported. \I:c also found the method of Lau rt al.’ to give erroneous results, probably because of the low pH used for liberation of the vitamin from serum protein and the omission of cyanide. II. The best precision and vc~jwoducibilifv of the metliod was achieved Lvlien the added amount of labelled I& was appro&ately equal to the mean (quantit! of IS,, present in normal samples. A larger addition of labelled I&, decreases the sensitivity of the method, nhereas a smaller quantity also decreases tlie reliability b~,r lowering the radioactivity to be measured. The quality of the labelled vitamin K 12 is of great importance for the resu1t.s. Fig. 1 illustrates curves of titration of 11; with two different commercial preparations of cobalamin. With the preparation used by us, a nearly complete absorption was achieved, \\hereas 1 I”,, of the radioactivity remained fret, when the other prclxration was used, even with a large excess of I I;. III. For the s/mijic hiudiu, 17of R,, xve have chosen intrinsic factor, since tlris protein is commercially available, and since it gives a stable and stoichiomctri(~ coupling with the vitamin. Sormal serum, used by Frenkel et nd.j, as well as myeloic serum”, are binders containing several proteins with affinity to the vitamin. IF is unstable in diluted solutionH, but this difficult\- is avoided by storing the stock solution in the frozen state, and making up a working solution for each series of determinations. The quality of tile IF is of paramount importance. The presence of other proteins may be of unpredictable influence on the binding stabilit!, and capacity, and different commercial preparations have different stability. The oncwe now use lias given repro&cible results wlien strict]\, adhering to the standard proccdurc. This is btrcau~ me carefullv centrifuge the stock solution and discard an\. undissolved residue, ii procedure ethic11 seems to have been neglected by previoux authors. \I’hen and if a crystalline preparation of IF will be available, this will lx-ol~al~l~~be preferable. IV. 1;or S?/!W&~lZ Of‘fJWfYfY & from that bound to intrinsic factor, our motlification of the 12chlm-g1 mctllod as modified by Sterling c? nl.” proved to 1~ con\:enicwt, and to gi\e I-eproducible results. Tllc loss of m”,, of tlrc activit\. b17 I nltraiiltraticm througlr tlw dialysis tubing ih csplained 13!- tlw fact that tllci tubing ahorbs sonitwater wlicii it is iiioistcwd. This water dilutes the ultrafiltrate lx m”,, of its voluine, since it constitutes about 200 mg of the I ml \-olume of filtrate. ‘171~~ reproducibilit~~ of tlie radioactivity of the filtrate from standard solutions, as \vell as tlrat from struni samples, dcmonstratcs that tlie degree of dilution is constant, and that coiilpariso~l of sample values with standard values gives correct results. In agreciiicnt \vitli 1iot1icn1~er~*, we obtainctl unrc~liable results xvhrn uGiig absorption of the free I:,, to active charcoal, as used I)>- I,au r? lr1.’ and by otlrcrs!‘~“‘. I-. In tlie prcsent iiictliod, the i~itauzill coucc~7ztvatio,z in tire sample c-allnot mail!, be calculated according to the formula of Rittenbcrg it nl. II because of tire dilution of the ultrafiltratc by water from the dialysis tubing. Howcvcr, comparison of tlrc activitlr with a standard curve, as described in the nictliod, was found convenient. Tlie present method is easy to perform, but recluircs facilities for rcfrigeratctl centrifuging and for detrrmitiation of gam~na radiation. Tt is \vcll suitctl fcor dctc>rmination of vitamin IS,, in series of I0 to 40 samples. .\ tecllnician can easilv anal\-sc 50 samples a dav.
VITAMIN
B,,
DETERMINATION