Clinica Chimica Acta, 203 (1991) 403,404 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009-8981/91/$03.50
403
CCA 05082
Letter
to the Editor
Isoelectric focusing of neutrophil alkaline phosphatase in trisomy 21 pregnancies (Received 13 May 1991, accepted 26 July 1991)
Dear Editor, Recent studies on neutrophil alkaline phosphatase CAP, EC: 3.1.3.1) from women with trisomy 21-affected fetuses assume that this protein phosphatase has biochemical and cytochemical properties distinct from that usually found in normal pregnancy [l-4]. The differential heat sensitivity of neutrophil AP in normal and pregnant women carrying trisomy 21 fetuses was one of the most relevant features. These first data led us to investigate isoelectrofocusing (IEF) patterns in both groups using a previously reported technique [5,6] with new improvements. Briefly, IEF analysis also confirmed a greater heat stability of neutrophil AP in trisomy 21 pregnancy. Karyotype determination were performed on fetal cells grown from amniotic fluid [7] of twelve women studied with high-risk pregnancies. In each case a free trisomy was detected. Neutrophil isolation from venous blood, AP extraction, assay and staining procedures have been reported [5,6]. When necessary, dialyzed extracts were concentrated by centrifugation with Centrisart tubes to get comparative AP activities in samples analyzed. A portion of each preparation was heated at 56 o C for 15 min prior to focusing. Some technical changes have been worked out. Cathode and anode electrolyte concentrations have been decreased respectively to 0.020 M NaOH and 0.005 M H,SO,. Upper limits of electric parameters were: 1200 V, 10 mA, 6 W for 1 h 20 min. The IEF patterns of neutrophil AP in cases considered are shown in Fig. 1. In absence of heat treatment (b,, ci, d,, e,, fi> the enzymatic activity electrofocused into two main isoform groups (pZ 7.0-6.1 and 4.9-4.4). Improvement in the resolution of AP patterns enables the separation of 5 or 6 bands with pZ 7.0, 6.8, 6.7, 6.5, 6.4, 6.1 and 3 or 4 more anodic bands with pZ 4.9, 4.7, 4.6, 4.4. As previously observed, the anodic component was very unstable after storage at - 80 ’ C (fl). All our experiment data support the view that the variable number of isoform bands, in particular for the anodic group, were not related to gestational stage, nor to fetal abnormality. Therefore, in spite of changes found in AP
404
As previously observed, the anodic component was very unstable after storage at - 80 o C (fl). All our experiment data support the view that the variable number of isoform bands, in particular for the anodic group, were not related to gestational stage, nor to fetal abnormality. Therefore, in spite of changes found in AP antigenicity [1,2] in the women with fetal trisomy 21 syndrome, focusing patterns appeared roughly similar to those of controls although a diffuse zone of AP activity near to the cathode was often stronger in trisomy 21 pregnancies (d,, e,, f,). It was independent of different protein loadings or enzymatic activities and of pregnancy trimesters, since in the normal pregnant women the IEF pattern remained unchanged during the gestational time course. In contrast, incubation at 56°C for 15 min resulted in marked differences in IEF diagrams. While the enzymatic activity of extracts from normal pregnant woman practically disappeared (c,) neutrophil AP isoenzymes from women with a trisomic 21 fetus cd,, e,, f,) displayed a higher thermostability. It can be noticed that freezing diminished enzyme resistance to heat denaturation (f,). This fact is consistent with the need to compare freshly prepared extracts. Normal control AP (b,) presented an intermediate thermostability. These results agree well with previously reported cytochemical and biochemical findings [ 11. Another point of interest must be considered. Starting from the present technique on a thin layer agarose gel, development of a preparative method is now conceivable. This new step will allow further determination on biochemical and immunological characteristics of purified enzymes. AndrCe Brisson-Lougarre ‘, Henri Vergnes 2, Jean Grozdea 2, G. Bourrouillou 3 and P. Colombies 3 ’ Luboratoire d’lmmunopthologie r&ale, Fact&P de Mdecine, Toulouse, ’ Sercice universitaire d’H6matologie, Laboratoire d’enzymologie du granulocyte, FacultP de Midecine de Toulouse-Rangueil, and 3 Laboratoire Central d’H6matologie et de Gb&que, Toulouse (Fiance)
Reference 1 Vergnes H, Grozdea J, Bourrouillou G, Brisson-Lougarre A, Fournie A, Colombies P. Biochemical and immunological properties of neutrophil alkaline phosphatases in normal pregnant women and in trisomy 21 pregnancies at the same gestational age (21/22 weeks). Biol Res Pregnancy 1986,7:145-148.
Correspondence to: H. Vergnes, Laboratoire d’HCmatologie - Service Universitaire, 31052 Toulouse Cedex, France.
CHU Rahgueil,
403
CCA 05082
Letter
to the Editor
Isoelectric focusing of neutrophil alkaline phosphatase in trisomy 21 pregnancies (Received 13 May 1991, accepted 26 July 1991)
Dear Editor, Recent studies on neutrophil alkaline phosphatase CAP, EC: 3.1.3.1) from women with trisomy 21-affected fetuses assume that this protein phosphatase has biochemical and cytochemical properties distinct from that usually found in normal pregnancy [l-4]. The differential heat sensitivity of neutrophil AP in normal and pregnant women carrying trisomy 21 fetuses was one of the most relevant features. These first data led us to investigate isoelectrofocusing (IEF) patterns in both groups using a previously reported technique [5,6] with new improvements. Briefly, IEF analysis also confirmed a greater heat stability of neutrophil AP in trisomy 21 pregnancy. Karyotype determination were performed on fetal cells grown from amniotic fluid [7] of twelve women studied with high-risk pregnancies. In each case a free trisomy was detected. Neutrophil isolation from venous blood, AP extraction, assay and staining procedures have been reported [5,6]. When necessary, dialyzed extracts were concentrated by centrifugation with Centrisart tubes to get comparative AP activities in samples analyzed. A portion of each preparation was heated at 56 o C for 15 min prior to focusing. Some technical changes have been worked out. Cathode and anode electrolyte concentrations have been decreased respectively to 0.020 M NaOH and 0.005 M H,SO,. Upper limits of electric parameters were: 1200 V, 10 mA, 6 W for 1 h 20 min. The IEF patterns of neutrophil AP in cases considered are shown in Fig. 1. In absence of heat treatment (b,, ci, d,, e,, fi> the enzymatic activity electrofocused into two main isoform groups (pZ 7.0-6.1 and 4.9-4.4). Improvement in the resolution of AP patterns enables the separation of 5 or 6 bands with pZ 7.0, 6.8, 6.7, 6.5, 6.4, 6.1 and 3 or 4 more anodic bands with pZ 4.9, 4.7, 4.6, 4.4. As previously observed, the anodic component was very unstable after storage at - 80 ’ C (fl). All our experiment data support the view that the variable number of isoform bands, in particular for the anodic group, were not related to gestational stage, nor to fetal abnormality. Therefore, in spite of changes found in AP
404
As previously observed, the anodic component was very unstable after storage at - 80 o C (fl). All our experiment data support the view that the variable number of isoform bands, in particular for the anodic group, were not related to gestational stage, nor to fetal abnormality. Therefore, in spite of changes found in AP antigenicity [1,2] in the women with fetal trisomy 21 syndrome, focusing patterns appeared roughly similar to those of controls although a diffuse zone of AP activity near to the cathode was often stronger in trisomy 21 pregnancies (d,, e,, f,). It was independent of different protein loadings or enzymatic activities and of pregnancy trimesters, since in the normal pregnant women the IEF pattern remained unchanged during the gestational time course. In contrast, incubation at 56°C for 15 min resulted in marked differences in IEF diagrams. While the enzymatic activity of extracts from normal pregnant woman practically disappeared (c,) neutrophil AP isoenzymes from women with a trisomic 21 fetus cd,, e,, f,) displayed a higher thermostability. It can be noticed that freezing diminished enzyme resistance to heat denaturation (f,). This fact is consistent with the need to compare freshly prepared extracts. Normal control AP (b,) presented an intermediate thermostability. These results agree well with previously reported cytochemical and biochemical findings [ 11. Another point of interest must be considered. Starting from the present technique on a thin layer agarose gel, development of a preparative method is now conceivable. This new step will allow further determination on biochemical and immunological characteristics of purified enzymes. AndrCe Brisson-Lougarre ‘, Henri Vergnes 2, Jean Grozdea 2, G. Bourrouillou 3 and P. Colombies 3 ’ Luboratoire d’lmmunopthologie r&ale, Fact&P de Mdecine, Toulouse, ’ Sercice universitaire d’H6matologie, Laboratoire d’enzymologie du granulocyte, FacultP de Midecine de Toulouse-Rangueil, and 3 Laboratoire Central d’H6matologie et de Gb&que, Toulouse (Fiance)
Reference 1 Vergnes H, Grozdea J, Bourrouillou G, Brisson-Lougarre A, Fournie A, Colombies P. Biochemical and immunological properties of neutrophil alkaline phosphatases in normal pregnant women and in trisomy 21 pregnancies at the same gestational age (21/22 weeks). Biol Res Pregnancy 1986,7:145-148.
Correspondence to: H. Vergnes, Laboratoire d’HCmatologie - Service Universitaire, 31052 Toulouse Cedex, France.
CHU Rahgueil,
