239
Clinica Chimica Acta, 93 (1979) 239-244 0 ElsevierlNorth-Holland Biomedical Press
CCA 10095
FORMATION OF A VITAMIN AT ALKALINE pH
V. LEE-OWN
*, A.E. BOLTON
B-12-SERUM
COMPLEX
ON HEATING
and P.J. CARR
Medical College of St. Bartholomew k Hospital, Immunoassay Research and Development Unit, 48-50 Bartholomew Close, West Smithfield, London EC1 (U.K.) (Received
October
4th, 1978)
Summary
The binding of vitamin B-12 to serum proteins during heating at alkaline pH was investigated by gel filtration of serum supplemented with cyan~[~‘Co]cobalamin. Heating for 5 min at 100°C destroyed most of the vitamin B-12 binding activity of serum but, with further heating, the vitamin B-12 became incorporated into a complex that did not correspond in molecular size to the original vitamin B-12 binding proteins. Radioassay of vitamin ,B-12 in heated serum showed correspondingly first an increase then a progressive decrease in the apparent vitamin B-12 level suggesting that, on heating, vitamin B-12 was initially released then subsequently complexed by the serum. The formation of complexed vitamin B-12 was abolished by the presence of the reducing agent dithiothreitol during the heating step.
Introduction
Circulating vitamin B-12 (B12) is bound to at least three proteins, designated transcobalamins I, II, and III [l]. The commonly used radioassays for B12 incorporate a heating step at 100°C to destroy these endogenous binding proteins. Such assays are generally carried out at acid pH [2] but, with the recent introduction of simultaneous assays for serum B12 and folate, a pH of 9..3 has been used [ 31. Using a radioassay for B12 and gel filtration we have investigated the release of B12 from endogenous serum binders on heating at 100°C under alkaline conditions.
* To whom correspondence should be addressed.
Materials and methods Reagents
Sephadex G-25 (medium) and G-200 were obtained from Pharmacia (GB) Ltd., cyano[57Co]cobalamin (CT2) from The Radiochemical Centre, Amersham, U.K., uniabelled cy~ocob~~in (Cyt~en) from Glaxo Laboratories, bovine serum albumin (BSA) from Armour Pharmaceutical Co. Ltd., dithiothreitol (DTT), human albumin and human y-globulin from Sigma (London) Chemical Co. All other reagents were Analar or equivalent in grade. Heating of serum Serum (50 pi) from
a normal male volunteer was diluted with 250 ~1 of 50 mmol/l sodium borate buffer pH 9.3 cont~ning 0.15 mol/l NaCl, 0.02% KCN and about 50 pg of cyano~“Co]cob~~in. Where required, DTT was incorporated into the borate buffer at a concentration of 2 mg/ml. The mixtures were heated in a boiling water bath at 100°C allowed to cool to room temperature and further diluted with 250 ~1 of 50 mmol/l sodium borate buffer pH 9.3 containing 0.15 mol/l NaCl, 1 mg/ml BSA and 0.03% sodium azide. Gel fiitrat~on
The incubation mixtures were chromato~aphed on columns of Sephadex G-25 (0.55 X 30 cm) eluted at room temperature with 0.15 mol/l NaCl containing 1 mgfml BSA and 0.03% sodium azide. Those fractions which eluted at the void volume of the Sephadex G-25 columns and which contained cyano[57Co]cobalamin were further chromatographed on columns of Sephadex G-200 (1 X 60 cm). The columns were eluted at room temperature in the dark with 50 mmol/l sodium borate buffer pH 9.3 cont~ning 0.15 mol/l NaCl and 1 mgfml BSA. Radioassay
for Bl.2
The B12 radioassay was a modification of the method of Kubiatowicz et al. [ 43 and utilized a heating step carried out at pH 9.3 to denature the endogenous binding proteins of serum samples. Cyano[S7Co]cobalamin was added to the samples after the denaturation stage and the binding agent used was toadfish (Opsunus tau) serum. The protein-bound B12 was separated from the free fraction using a Dextran-coated charcoal system. Results Gel filtration
Serum was incubated with cyano[57Co]cobalamin to act as a marker for endogenous B12. Chromatography of serum samples on Sephadex G-25 clearly separated the free and bound cyano[57Cofcobalamin, the bound material appearing at the void volume of the column (Fig. 1). With unheated serum about 85% of the cyanoj S7Co]cobalamin eluted in the protein-bound fraction, corresponding to that B12 bound by serum transcobalamins (Fig. 1A). After heating at 100°C for 5-10 min most of the cyano[s7Co]cobalamin was eluted in the free fraction showing that the endogenous binders had become denatured
241
D
5 FRACTION
10
15
NUMBER
Fig. 1. Elution from Sephadex G-25 of cyano[ 57Co]cohalamin after incubation with serum. (A) Unheated serum and label incubated at room temperature. (B) Serum and label heated at 1OO’C for 10 min. (C) Serum and label heated at 100°C for 30 min. (D) Label heated in buffer at 1OO’C. 30 min. O-----O, heated in the absence of 2 mg/ml DTT; m---4, heated in the presence of 2 mg/ml DTT.
and the B12 released (Fig. 1B). With further heating there was a shift of radioactivity back to the void volume fraction indicating the incorporation of the B12 into a high molecular weight complex (Fig. 1C). Cyano[S7Co]cobalamin heated in the absence of serum did not form a complex that was excluded from Sephadex G-25 (Fig. 1D). When cyano[“Co]cobalamin was added to serum that had been heated previously, or was heated in the presence of a solution of 40 g/l human albumin or 30 g/l human y-globulin in place of serum, no complex formation could be detected. When DTT was present during the heating step no high molecular weight complex was detected (Fig. 1C). The molecular size-of the B12 complex formed with serum was characterized by chromatography on Sephadex G-200 (Fig. 2). The distribution of proteinbound cyano[ “Colcobalamin in unheated serum corresponded with previously reported results [ 51. However, the radioactivity in samples subjected to prolonged heating in the presence of serum eluted as a high molecular weight species at the void volume of the G-200 column. This material did not correspond in molecular weight to the known serum transcobalamins (Fig. 2). Radioassay results The radioassay had a working range of 100-2000 pg/ml. The within-assay coefficient of variation (C-V.) was. 5% and the between assay C.V. 9.6%. Dilu-
20
15
25
20 ELUATE
40
35
30
VOLUME
Cm,,
Fig. 2. Elution from Sephadex G-200 of cuano[ 5’Colcobalamin after incubation with serum. Vg, void volume of the column determined using Blue Dextran 2000. AA, unheated serum and label after incubation at room temperature, .A, material excluded from Sephadex G-25 after chromatography of label and serum heated at 1OO’C for 30 min (Fig. 1C).
TABLE
I
THE EFFECT
OF HEATING
AT 100°C
ON THE APPARENT
VITAMIN
B-12 CONCENTRATION
OF
SERUM Serum was heated in buffer the radioassay procedure. Time of heating serum (min)
with or without
Apparent
2 mg/ml
B12 concentration
+DTT
DTT and subsequently
assayed for vitamin
@g/ml) No DTT
Serum A
Serum B
Serum A
Serum B
5 10
Clinica Chimica Acta, 93 (1979) 239-244 0 ElsevierlNorth-Holland Biomedical Press
CCA 10095
FORMATION OF A VITAMIN AT ALKALINE pH
V. LEE-OWN
*, A.E. BOLTON
B-12-SERUM
COMPLEX
ON HEATING
and P.J. CARR
Medical College of St. Bartholomew k Hospital, Immunoassay Research and Development Unit, 48-50 Bartholomew Close, West Smithfield, London EC1 (U.K.) (Received
October
4th, 1978)
Summary
The binding of vitamin B-12 to serum proteins during heating at alkaline pH was investigated by gel filtration of serum supplemented with cyan~[~‘Co]cobalamin. Heating for 5 min at 100°C destroyed most of the vitamin B-12 binding activity of serum but, with further heating, the vitamin B-12 became incorporated into a complex that did not correspond in molecular size to the original vitamin B-12 binding proteins. Radioassay of vitamin ,B-12 in heated serum showed correspondingly first an increase then a progressive decrease in the apparent vitamin B-12 level suggesting that, on heating, vitamin B-12 was initially released then subsequently complexed by the serum. The formation of complexed vitamin B-12 was abolished by the presence of the reducing agent dithiothreitol during the heating step.
Introduction
Circulating vitamin B-12 (B12) is bound to at least three proteins, designated transcobalamins I, II, and III [l]. The commonly used radioassays for B12 incorporate a heating step at 100°C to destroy these endogenous binding proteins. Such assays are generally carried out at acid pH [2] but, with the recent introduction of simultaneous assays for serum B12 and folate, a pH of 9..3 has been used [ 31. Using a radioassay for B12 and gel filtration we have investigated the release of B12 from endogenous serum binders on heating at 100°C under alkaline conditions.
* To whom correspondence should be addressed.
Materials and methods Reagents
Sephadex G-25 (medium) and G-200 were obtained from Pharmacia (GB) Ltd., cyano[57Co]cobalamin (CT2) from The Radiochemical Centre, Amersham, U.K., uniabelled cy~ocob~~in (Cyt~en) from Glaxo Laboratories, bovine serum albumin (BSA) from Armour Pharmaceutical Co. Ltd., dithiothreitol (DTT), human albumin and human y-globulin from Sigma (London) Chemical Co. All other reagents were Analar or equivalent in grade. Heating of serum Serum (50 pi) from
a normal male volunteer was diluted with 250 ~1 of 50 mmol/l sodium borate buffer pH 9.3 cont~ning 0.15 mol/l NaCl, 0.02% KCN and about 50 pg of cyano~“Co]cob~~in. Where required, DTT was incorporated into the borate buffer at a concentration of 2 mg/ml. The mixtures were heated in a boiling water bath at 100°C allowed to cool to room temperature and further diluted with 250 ~1 of 50 mmol/l sodium borate buffer pH 9.3 containing 0.15 mol/l NaCl, 1 mg/ml BSA and 0.03% sodium azide. Gel fiitrat~on
The incubation mixtures were chromato~aphed on columns of Sephadex G-25 (0.55 X 30 cm) eluted at room temperature with 0.15 mol/l NaCl containing 1 mgfml BSA and 0.03% sodium azide. Those fractions which eluted at the void volume of the Sephadex G-25 columns and which contained cyano[57Co]cobalamin were further chromatographed on columns of Sephadex G-200 (1 X 60 cm). The columns were eluted at room temperature in the dark with 50 mmol/l sodium borate buffer pH 9.3 cont~ning 0.15 mol/l NaCl and 1 mgfml BSA. Radioassay
for Bl.2
The B12 radioassay was a modification of the method of Kubiatowicz et al. [ 43 and utilized a heating step carried out at pH 9.3 to denature the endogenous binding proteins of serum samples. Cyano[S7Co]cobalamin was added to the samples after the denaturation stage and the binding agent used was toadfish (Opsunus tau) serum. The protein-bound B12 was separated from the free fraction using a Dextran-coated charcoal system. Results Gel filtration
Serum was incubated with cyano[57Co]cobalamin to act as a marker for endogenous B12. Chromatography of serum samples on Sephadex G-25 clearly separated the free and bound cyano[57Cofcobalamin, the bound material appearing at the void volume of the column (Fig. 1). With unheated serum about 85% of the cyanoj S7Co]cobalamin eluted in the protein-bound fraction, corresponding to that B12 bound by serum transcobalamins (Fig. 1A). After heating at 100°C for 5-10 min most of the cyano[s7Co]cobalamin was eluted in the free fraction showing that the endogenous binders had become denatured
241
D
5 FRACTION
10
15
NUMBER
Fig. 1. Elution from Sephadex G-25 of cyano[ 57Co]cohalamin after incubation with serum. (A) Unheated serum and label incubated at room temperature. (B) Serum and label heated at 1OO’C for 10 min. (C) Serum and label heated at 100°C for 30 min. (D) Label heated in buffer at 1OO’C. 30 min. O-----O, heated in the absence of 2 mg/ml DTT; m---4, heated in the presence of 2 mg/ml DTT.
and the B12 released (Fig. 1B). With further heating there was a shift of radioactivity back to the void volume fraction indicating the incorporation of the B12 into a high molecular weight complex (Fig. 1C). Cyano[S7Co]cobalamin heated in the absence of serum did not form a complex that was excluded from Sephadex G-25 (Fig. 1D). When cyano[“Co]cobalamin was added to serum that had been heated previously, or was heated in the presence of a solution of 40 g/l human albumin or 30 g/l human y-globulin in place of serum, no complex formation could be detected. When DTT was present during the heating step no high molecular weight complex was detected (Fig. 1C). The molecular size-of the B12 complex formed with serum was characterized by chromatography on Sephadex G-200 (Fig. 2). The distribution of proteinbound cyano[ “Colcobalamin in unheated serum corresponded with previously reported results [ 51. However, the radioactivity in samples subjected to prolonged heating in the presence of serum eluted as a high molecular weight species at the void volume of the G-200 column. This material did not correspond in molecular weight to the known serum transcobalamins (Fig. 2). Radioassay results The radioassay had a working range of 100-2000 pg/ml. The within-assay coefficient of variation (C-V.) was. 5% and the between assay C.V. 9.6%. Dilu-
20
15
25
20 ELUATE
40
35
30
VOLUME
Cm,,
Fig. 2. Elution from Sephadex G-200 of cuano[ 5’Colcobalamin after incubation with serum. Vg, void volume of the column determined using Blue Dextran 2000. AA, unheated serum and label after incubation at room temperature, .A, material excluded from Sephadex G-25 after chromatography of label and serum heated at 1OO’C for 30 min (Fig. 1C).
TABLE
I
THE EFFECT
OF HEATING
AT 100°C
ON THE APPARENT
VITAMIN
B-12 CONCENTRATION
OF
SERUM Serum was heated in buffer the radioassay procedure. Time of heating serum (min)
with or without
Apparent
2 mg/ml
B12 concentration
+DTT
DTT and subsequently
assayed for vitamin
@g/ml) No DTT
Serum A
Serum B
Serum A
Serum B
5 10
