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Vitamin B12-binding proteins in human amniotic fluid: An index to fetal maturity
COMMUNICATIONS IN BRIEF
:/ I
This section is suitable for reporting results of therapeutic trials, descriptions ot new procedures or instruments, and case reports which illustrate a principle. Reports should be limited to seven hundred words and two references. Use of an illustration or table requires a proportionate reduction in total words.
Vitamin B,,-binding proteins in human amniotic fluid: An index to fetal maturity N. Grossowicz, and Y. Beyth,
Ph.D., M.D.
Mala Jablonska,
Department of Bacteriology, The Hebrew University-Hadassah Medical School, and the Dqartment of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel
THE TRANSCOBALAMINS (TCs) are trace proteins found in human serum, which bind vitamin B,,. Moreover, one of the vitamin B,,-binding proteins, TC I, is present in human amniotic fluid in much higher concentrations than are found in serum.’ Several inherited disorders of defective TCs have been described, among them a recessively inherited deficiency of TC II, which results in megaloblastic anemia and failure to thrive.” We recently developed a simple, rapid, and sensitive assay for the simultaneous quantitation of the three TCs present in human serum and thus also of the unsaturated B,,-binding capacity.3 In the following, a preliminary report on the distribution of the TCs and the unsaturated B,,-binding capacity in human amniotic fluid during the second and third trimesters of pregnancy is given; from the report the potential uses of the TCs as indicators of fetal age and for the presumptive prenatal diagnosis of deficiency are suggested. A total of 142 amniocenteses in 125 gravid patients, obtained with their prior consent, were performed for the assessment of fetal maturity. The amniotic fluid samples were chilled immediately after they were obtained and centrifuged at 500 x g; the supernates were stored at -20” C until assayed.
George
M. Jaffin,
New
York,
New
available to N. G. Reprint requests: Nathan Grossowicz, of Bacteriology, The Hebrew School, Jerusalem, Israel.
York,
Ph.D., University-Hadassah
made
a grant
Department Medical
Our charged cellulose filtration procedure” was used for the quantitative assay of the three vitamin B,,binding proteins (TC I, TC 11, and TC III) and for calculation of the unsaturated B,,-binding capacity; only 20 ~1 of amniotic fluid was needed for the complete assay. The assay material consisted of the sample (20 11). borate buffer, pH 8.5, 100 pg of “‘Co-vitamin B,, of high specific activity in a total volume of 0.5 ml, and a stack of four cellulose-filter disks, a kind of minicolumn, 25 mm in diameter, placed in a Millipore-type filter-holder apparatus; the stack was composed of a single cellulose-nitrate filter (Schleicher and Schiill, Dassel. Germany) which was placed on top of three DE-81 filters (The Whatman Biochemicals Ltd., Maidstone, Kent, England). The reaction mixture was incubated for 30 minutes at 37” C, filtered through the filter stack, and washed with buffer for removal of unbound vitamin B12. The filter-bound radioactivity was determined separately on each filter-type with a gamma counter. The detailed procedure was described recently by us3 Usually 20 to 40 samples were assayed during 3 hours by a single technician. About 25% of the samples were reassayed after about 2 months with almost identical results, which attests to the stability of the TCs in the stored (-20” C) amniotic fluid. The unsaturated B,,-binding capacity was detectable in the amniotic fluid at about the seventeenth to eighteenth week of gestation and by the twentieth week had reached values found in sera of healthy subjects.” The B,, binding continued to increase rather sharply with increase of gestational age until values fivefold to tenfold higher than those of sera of healthy subjects” were reached at the thirty-fifth to fortieth week of gestation (Fig. 1). Thus, there was a twent.yfold to thirtyfold increase in the B,2-binding capacity from 300 and 285 ng of z7Co-vitamin B,, per liter during the sixteenth and seventeenth weeks to 9,350 and 6,150 rig/L at the thirty-ninth and fortieth weeks, respectively. Although the increase in the unsaturated B,%binding capacity appears linear, further samples of amniotic fluid between the twenty-fifth and thirty-third 331
332
Communications
in brief
June
Am. J. Obstet.
I
I
I
I
I
I
I
I
I
I
I
I
I
A
’
1. 1983
Gynecol.
-1 00
. a
9oocI~8000 3 7000 26000 -5000 8. ;4000 0
CD'3000 0
-*-
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6’‘* A d .N’
A
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I
I
"14 16 18 20 22
I
1
I
I
I
I
1
0 I
0 I
24 26 28 30 32 34 36 38 40
WEEKS
OF PREGNANCY
Fig. 1. Unsaturated B,,-binding capacity and TCs in amniotic fluid as a function of gestational A = unsaturated B,,-binding capacity; l = TC I; o = TC II: q = TC III.
weeks of gestation are required for confirmation of the present findings. The increase in TC I in the amniotic fluid at first paralleled the increase in unsaturated Blz binding. Following very low values at the sixteenth week to the eighteenth week (16% to 35% of the unsaturated Blz-binding capacity, TC I increased steeply to 78% during the twenty-fifth week, and then the B,,-binding capacity remained static or increased slightly. Values for TC II, on the other hand, were highest early in pregnancy (fifteenth and sixteenth weeks), amounting to about 60% of the Blz-binding capacity, when both the B,,-binding capacity and TC I showed the lowest values (Fig. 1). Thereafter, the TC II decreased steadily to a low value of 12% at the twenty-fourth week of gestation and remained low or even decreased further toward the end of gestation. Values for TC III did not change appreciably during the entire period, fiuctuating between 17% and 30% (mean = 23.3% 2 1.05%) of the Blz binding. The proportions of TCs in the anniotic fluid were similar to those in sera of healthy adult subjects only during very early gestation3 (sixteenth week: TC II, 57.5%; TC I, 17.1%; TC III, 25.4%). At the beginning of the third trimester of pregnancy, the amniotic fluid TC II decreased to low values, (2% to 12%) while TC I increased sharply (66% to 78%), contrary to findings in sera of healthy subjects. Concomitant with the increase in TC I, the vitamin Blz binding increased to values fivefold those found in sera of healthy subjects. Because the unsaturated Blz-binding capacity increased by twentyfold to thirtyfold in the interval from the fifteenth week of gestation to term, a greater change than is found with any enzymes thus far measured in
age,
the amniotic fluid, this parameter seems to prove of value as an index of fetal age and status. Further data on the vitamin Blz binding of amniotic fluid of both normal term infants and small-for-dates infants and of fluid associated with other disorders of the fetus will determine the usefulness of the measurement. The amniotic fluid TC II accounted for about 60% of B,,-binding capacity at the fifteenth to seventeenth week of gestation, decreasing steadily with increasing gestational age to very low values at the end of the second trimester. These findings may be useful in the prenatal diagnosis of the rare TC II deficiency in families at risk.’ Thus, profoundly low values at 15 to 18 weeks of gestation would indicate the prenatal diagnosis of the affected fetus. A subsequent determination on an amniotic fluid sample obtained 2 weeks later would be of confirmatory value. This test would, however, not apply in case of biologically inert TC II, a very rare abnormality, characterized by failure of transfer of vitamin BfZ to the tissues while not affecting the binding of the vitamin.’
REFERENCES
1. Stenman, U. H.: Amniotic tein.
Purification
and
fluid vitamin BIZ-binding
characterization
with
pro-
isoelectric
focusing and other techniques, Biochim. Biophys. Acra 349:173, 1974. 2. Haurani, F. I., Hall, C. A., and Rubin, R. N.: Megaloblastic anemia due to an abnormal transcobalamin II, Blood 48:9&l, 1976. 3. Selhub, J., Rachmilewitz, B., and Grossowicz, N.: Fractionation of serum transcobalamins on charged cellulose filters, Proc. Sot. Exp. Biol. Med. U. S. A. 15!2:161, 1976.
:/ I
This section is suitable for reporting results of therapeutic trials, descriptions ot new procedures or instruments, and case reports which illustrate a principle. Reports should be limited to seven hundred words and two references. Use of an illustration or table requires a proportionate reduction in total words.
Vitamin B,,-binding proteins in human amniotic fluid: An index to fetal maturity N. Grossowicz, and Y. Beyth,
Ph.D., M.D.
Mala Jablonska,
Department of Bacteriology, The Hebrew University-Hadassah Medical School, and the Dqartment of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel
THE TRANSCOBALAMINS (TCs) are trace proteins found in human serum, which bind vitamin B,,. Moreover, one of the vitamin B,,-binding proteins, TC I, is present in human amniotic fluid in much higher concentrations than are found in serum.’ Several inherited disorders of defective TCs have been described, among them a recessively inherited deficiency of TC II, which results in megaloblastic anemia and failure to thrive.” We recently developed a simple, rapid, and sensitive assay for the simultaneous quantitation of the three TCs present in human serum and thus also of the unsaturated B,,-binding capacity.3 In the following, a preliminary report on the distribution of the TCs and the unsaturated B,,-binding capacity in human amniotic fluid during the second and third trimesters of pregnancy is given; from the report the potential uses of the TCs as indicators of fetal age and for the presumptive prenatal diagnosis of deficiency are suggested. A total of 142 amniocenteses in 125 gravid patients, obtained with their prior consent, were performed for the assessment of fetal maturity. The amniotic fluid samples were chilled immediately after they were obtained and centrifuged at 500 x g; the supernates were stored at -20” C until assayed.
George
M. Jaffin,
New
York,
New
available to N. G. Reprint requests: Nathan Grossowicz, of Bacteriology, The Hebrew School, Jerusalem, Israel.
York,
Ph.D., University-Hadassah
made
a grant
Department Medical
Our charged cellulose filtration procedure” was used for the quantitative assay of the three vitamin B,,binding proteins (TC I, TC 11, and TC III) and for calculation of the unsaturated B,,-binding capacity; only 20 ~1 of amniotic fluid was needed for the complete assay. The assay material consisted of the sample (20 11). borate buffer, pH 8.5, 100 pg of “‘Co-vitamin B,, of high specific activity in a total volume of 0.5 ml, and a stack of four cellulose-filter disks, a kind of minicolumn, 25 mm in diameter, placed in a Millipore-type filter-holder apparatus; the stack was composed of a single cellulose-nitrate filter (Schleicher and Schiill, Dassel. Germany) which was placed on top of three DE-81 filters (The Whatman Biochemicals Ltd., Maidstone, Kent, England). The reaction mixture was incubated for 30 minutes at 37” C, filtered through the filter stack, and washed with buffer for removal of unbound vitamin B12. The filter-bound radioactivity was determined separately on each filter-type with a gamma counter. The detailed procedure was described recently by us3 Usually 20 to 40 samples were assayed during 3 hours by a single technician. About 25% of the samples were reassayed after about 2 months with almost identical results, which attests to the stability of the TCs in the stored (-20” C) amniotic fluid. The unsaturated B,,-binding capacity was detectable in the amniotic fluid at about the seventeenth to eighteenth week of gestation and by the twentieth week had reached values found in sera of healthy subjects.” The B,, binding continued to increase rather sharply with increase of gestational age until values fivefold to tenfold higher than those of sera of healthy subjects” were reached at the thirty-fifth to fortieth week of gestation (Fig. 1). Thus, there was a twent.yfold to thirtyfold increase in the B,2-binding capacity from 300 and 285 ng of z7Co-vitamin B,, per liter during the sixteenth and seventeenth weeks to 9,350 and 6,150 rig/L at the thirty-ninth and fortieth weeks, respectively. Although the increase in the unsaturated B,%binding capacity appears linear, further samples of amniotic fluid between the twenty-fifth and thirty-third 331
332
Communications
in brief
June
Am. J. Obstet.
I
I
I
I
I
I
I
I
I
I
I
I
I
A
’
1. 1983
Gynecol.
-1 00
. a
9oocI~8000 3 7000 26000 -5000 8. ;4000 0
CD'3000 0
-*-
g 2000
&.o-‘.
6’‘* A d .N’
A
0
/ 0 y’ ;-•-”
Id!!!
-.-.
0 -.-.n
-.o-.rl?_.-
\ /
1000 0
\ Jo .i \ ,,p’%.”
ICI1 o-o-
A.0 -II’
A--;
,A@-
A I
I
I
"14 16 18 20 22
I
1
I
I
I
I
1
0 I
0 I
24 26 28 30 32 34 36 38 40
WEEKS
OF PREGNANCY
Fig. 1. Unsaturated B,,-binding capacity and TCs in amniotic fluid as a function of gestational A = unsaturated B,,-binding capacity; l = TC I; o = TC II: q = TC III.
weeks of gestation are required for confirmation of the present findings. The increase in TC I in the amniotic fluid at first paralleled the increase in unsaturated Blz binding. Following very low values at the sixteenth week to the eighteenth week (16% to 35% of the unsaturated Blz-binding capacity, TC I increased steeply to 78% during the twenty-fifth week, and then the B,,-binding capacity remained static or increased slightly. Values for TC II, on the other hand, were highest early in pregnancy (fifteenth and sixteenth weeks), amounting to about 60% of the Blz-binding capacity, when both the B,,-binding capacity and TC I showed the lowest values (Fig. 1). Thereafter, the TC II decreased steadily to a low value of 12% at the twenty-fourth week of gestation and remained low or even decreased further toward the end of gestation. Values for TC III did not change appreciably during the entire period, fiuctuating between 17% and 30% (mean = 23.3% 2 1.05%) of the Blz binding. The proportions of TCs in the anniotic fluid were similar to those in sera of healthy adult subjects only during very early gestation3 (sixteenth week: TC II, 57.5%; TC I, 17.1%; TC III, 25.4%). At the beginning of the third trimester of pregnancy, the amniotic fluid TC II decreased to low values, (2% to 12%) while TC I increased sharply (66% to 78%), contrary to findings in sera of healthy subjects. Concomitant with the increase in TC I, the vitamin Blz binding increased to values fivefold those found in sera of healthy subjects. Because the unsaturated Blz-binding capacity increased by twentyfold to thirtyfold in the interval from the fifteenth week of gestation to term, a greater change than is found with any enzymes thus far measured in
age,
the amniotic fluid, this parameter seems to prove of value as an index of fetal age and status. Further data on the vitamin Blz binding of amniotic fluid of both normal term infants and small-for-dates infants and of fluid associated with other disorders of the fetus will determine the usefulness of the measurement. The amniotic fluid TC II accounted for about 60% of B,,-binding capacity at the fifteenth to seventeenth week of gestation, decreasing steadily with increasing gestational age to very low values at the end of the second trimester. These findings may be useful in the prenatal diagnosis of the rare TC II deficiency in families at risk.’ Thus, profoundly low values at 15 to 18 weeks of gestation would indicate the prenatal diagnosis of the affected fetus. A subsequent determination on an amniotic fluid sample obtained 2 weeks later would be of confirmatory value. This test would, however, not apply in case of biologically inert TC II, a very rare abnormality, characterized by failure of transfer of vitamin BfZ to the tissues while not affecting the binding of the vitamin.’
REFERENCES
1. Stenman, U. H.: Amniotic tein.
Purification
and
fluid vitamin BIZ-binding
characterization
with
pro-
isoelectric
focusing and other techniques, Biochim. Biophys. Acra 349:173, 1974. 2. Haurani, F. I., Hall, C. A., and Rubin, R. N.: Megaloblastic anemia due to an abnormal transcobalamin II, Blood 48:9&l, 1976. 3. Selhub, J., Rachmilewitz, B., and Grossowicz, N.: Fractionation of serum transcobalamins on charged cellulose filters, Proc. Sot. Exp. Biol. Med. U. S. A. 15!2:161, 1976.