- Email: [email protected]
A time-study analysis to evaluate albumin carbamylation in vitro
POSTER ABSTRACTS
phosphorylation was accompanied by an increase in the affinity of PDE for CaM and Ca2.. Analysis of the complex regulatory properties of PDE has led to the suggestion that fluxes of cAMP and Ca2÷ during cell activation are closely coupled, and that PDE plays a key role in the signalcoupling phenomenon. (Supported by the Heart and Stroke Foundation of Saskatchewan).
Comparison of the predictive performance of biochemical markers and mean arterial pressure f o r p r e - e c l a m p s i a Mass~, J., Forest, J-C., Moutquin, J.M., Marcoux, S., Brideau, N.A. and B61anger, M. D~partements de Biochimie, Obst6trique-Gyn6cologie, M6Aecine Sociale et Pr6ventive, Facult6 de M6Aecine, Universit6 Laval, Cit6 Universitaire, Qu6bec, G1K 7P4, Canada We prospectively evaluated the predictive performance of several potential biochemical markers of pre-eclampsia readily available in the clinical setting. We measured these markers (n = 27) on three occasions during pregnancy (814, 15-24, 25-34 weeks of gestation) in 1,366 nulliparous women. After delivery, women were classified into three categories: normal, pre-eclampsia, transient hypertension. The predictive performance of the tests, either used alone or in combination (stepwise multiple logistic regression), was assessed and compared to that of the mean arterial pressure. Pre-eelampsia developed in 109 of the pregnant women. Significant difference in the mean value of the tests between the normal and pre-eclamptic women, after Bonferroni correction, was only observed for three biochemical variables: progesterone, mean corpuscular volume and haptoglobin. Multiple logistic regression models were developed using the biochemical and clinical variables. Their predictive performance was compared with that of the mean arterial pressure using a cut-off value at a given specificity of 80%. The sensitivity, positive and negative predictive values for mean arterial pressure were respectively 46.6%, 23.5%, 92.0% at the second visit and 52.5%, 26.0% and 92.6% at the third visit. A logistic model for the second visit that included the mean arterial pressureshowed a sensitivity of 57.1%, a positive predictive value of 26.9% and a negative predictive value of 93.7%. For the model developed for the third visit, the corresponding values were 66.1%, 30.0% and 94.6%. Combination of the tests in a stepwise multiple regression model offered little advantages over the single use of mean arterial pressure.
CLINICALBIOCHEMISTRY,VOLUME 26, APRIL 1993
-ffq
A preliminary prospective evaluation of
b i o c h e m i c a l m a r k e r s f o r fetal t r i s o m i e s Forest, J-C., Mass6, J., Rousseau, F., Moutquin, J.M., Brideau, N.A., B61anger, M. and Bastide, A. D6partements de Biochimie et Obst~trique-Gyn6cologie, Facult~ de M6Aecine, Universit6 Laval, Cit6 Universitaire, Qu6bec, G1K 7P4, Canada We are currently measuring three markers (alphafetoprotein, estriol, choriogonadotropin) in pregnant women of all ages to determine their value in screening for fetal trisomies. Our study population consists of 9,000 women who attended the perinatal clinics of six hospitals and had signed a consent form. A blood specimen was obtained between 10 and 19 weeks gestation and the serum was stored at -20°C. Alphafetoprotein and choriogonadotropin were rrleasured with Enzymun-Test R enzymeimmunoassays (Boehringer Mannheim Canada). Unconjugated estriol was assayed with an ultrasensitive radioimmunometric assay (DSL Canada). Random samples from unaffected pregnancies were used to determine the distribution of results at each week. The maternal agespecific risk (a priori odds) was determined using published results (Br J Obstet G~aecol 1987; 94:387-402). The woman's multiples of median for each marker were used to calculate a likelihood ratio using the distribution parameters derived from our population. The posteriori odds were obtained by multiplying the a priori odds by the likelihood ratios. The results were considered positive if the calculated risk was equal to or higher than that of women of 35 years. Thirteen cases of singleton trisomy 21 occurred. Using age in combination with the three biochemical markers, 53.8% of these cases have been detected. No cases that happened in women 35 years or older would have been missed. These results confirm the sensitivity published by other groups. The application of this screening strategy in the older women does not appear to affect detection of a trisomic foetus while obviating a number of unnecessary amniocenteses. II
METHOD AND INSTRUMENT EVALUATION
[]
A t i m e - s t u d y a n a l y s i s to e v a l u a t e a l b u m i n c a r b a m y l a t i o n in vitro Balion, C.M., Caines, P.S.M., Draisey, T.F. and Thibert, g.J. Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, N9B 3P4, Canada
135
37TH ANNUAL CONFERENCE OF THE CANADIAN SOCIETY OF CLINICAL CHEMISTS
Carbamylation is a rapid, nonenzymatic reaction occurring between isocyanic acid, the spontaneous dissociation product of urea, and proteins. This process is increased in vivo in chronic renal failure patients, who have high urea levels. Human albumin (40 mg/mL) was incubated at room temperature with 0.1M sodium cyanate in 0.1M sodium phosphate buffer, pH 7.4, for four days. During the incubation, aliquots were removed and the albumin separated from excess cyanate by means of a P-6DG exclusion column. The desalted albumin samples were applied to an ultrathin polyacrylamide gel (Bio-Lyte 4/6) and focused using a Bio-Rad ° Mini IEF Cell. The degree of carbamylation was measured colorimetrically using diacetyl monoxime and expressed as absorbance units/mg albumin. The pI of the carbamylated albumin decreased from 4.9 (native albumin) to 4,2 after 40 h of incubation. Similarily, the number of carbamyl groups increased in proportion to the change in pI. These results indicate that the carbamylation process can be easily detected in vitro and that a saturating level of carbamylation occurs. The techniques employed in this study may be useful in evaluating the extent of carbamylation in patients with chronic renal failure.
D
Detection of PCR-amplified chronic myelogenous l e u k e m i a - s p e c i f i c m R N A sequences by i m m u n o f l u o r o m e t r y
Bortolin, S. and Christopoulos, T.K. Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Street, Windsor, Ontario, N9B 3P4, Canada The Philadelphia chromosome (Ph) is present in >95% of chronic myelogenous leukemia (CML) patients. It involves a reciprocal translocation, which fuses the ABL protooncogene to the BCR gene. Expression of the fusion gene results in a leukemia-specific BCR-ABL mRNA. To facilitate the diagnosis and monitoring of CML, we developed a highly sensitive, nonisotopic microtiter platebased assay for leukemia-specific mRNA in leukocytes. K562, a Ph-positive cell line is used as a positive control. mRNA is isolated from 103-106 leukocytes. A cDNA copy is synthesized by reverse transcription. Then, the diagnostically useful cDNA sequence is amplified by nested polymerase chain reaction (PCR). PCR I uses unlabeUed 21mer and 24met primers homologous to BCR and ABL, respectively. PCR II employs 22mer primers, 5'end-labelled with digoxigenin (DIG) and biotin. PCR I is performed for 25 cycles followed by 15 cycles of PCR II. The 200 bp amplified product carries biotin at one end and DIG at the
136
other. The reaction mixture is transferred to streptavidincoated microtiter wells where the PCR product is captured. Afterwards, alkaline phosphatase-labelled antidigoxigenin antibody is added. Fluorosalicylphosphate is used as substrate. The released fluorosalicylate, forms highly fluorescent complexes with Tb3+-EDTA. The fluorescence is measured with a time-resolved fluorometer. Amplified product corresponding to a single leukemic cell can be detected. Only Ph-positive cells give a double-labelled product. The whole procedure takes about 6 h. The proposed method avoids time-consuming steps such as electrophoresis and Southern blot and the use of radioisotopes.
Fq
Quantitative polymerase chain reaction analysis of pathogenic DNA sequence using an internal DNA sequence standard constructed by recombinant DNA methodology
Chan, A., Krajden, M. and Diamandis, E.P. Department of Clinical Biochemistry and Microbiology, The Toronto Hospital, 200 Elizabeth Street, Toronto, Ontario, M5G 2CA, Canada The successful development of sensitive and reliable quantitative assays for polymerase chain reaction (PCR) products using High Performance Liquid Chromatography (HPLC) have opened up new research and clinical applications that promote the understanding of pathology, pharmaco-toxicology, therapeutic management and diagnosis ofvarions diseases. However, it has been difficult to correlate the amount of PCR products output to the amount of DNA targets input which should be the more meaningful data for clinical applications. There are multiple factors leading to this analytical problem, such as the reproducibility of PCR thermoeycling runs, the integrity of reagents used, the PCR conditions and kinetics, the sample preparation methodologies and matrix interference, etc. By constructing a recombinant cytomegalovirus (CMV) control target plasmid which contained a modified CMV AD169 sequence that shared the PCR primer sequence of native CMV AD169 and co-amplified both the unknown and control plasmid targets, we were able to by-pass some of the kinetic PCR problems and to quantify native CMV AD169 targets. PCR co-amplification of the CMV control and CMV native plasmid targets resulted in two distinctively separable PCR products of 362 bp and 152 bp respectively, with a 15 rain HPLC run. During PCR coamplification there was an overall decrease as well as competition in the synthesis of the PCR products from both plasmid targets as compared to non-co-amplified
CLINICAL BIOCHEMISTRY, VOLUME 26, APRIL 1993
phosphorylation was accompanied by an increase in the affinity of PDE for CaM and Ca2.. Analysis of the complex regulatory properties of PDE has led to the suggestion that fluxes of cAMP and Ca2÷ during cell activation are closely coupled, and that PDE plays a key role in the signalcoupling phenomenon. (Supported by the Heart and Stroke Foundation of Saskatchewan).
Comparison of the predictive performance of biochemical markers and mean arterial pressure f o r p r e - e c l a m p s i a Mass~, J., Forest, J-C., Moutquin, J.M., Marcoux, S., Brideau, N.A. and B61anger, M. D~partements de Biochimie, Obst6trique-Gyn6cologie, M6Aecine Sociale et Pr6ventive, Facult6 de M6Aecine, Universit6 Laval, Cit6 Universitaire, Qu6bec, G1K 7P4, Canada We prospectively evaluated the predictive performance of several potential biochemical markers of pre-eclampsia readily available in the clinical setting. We measured these markers (n = 27) on three occasions during pregnancy (814, 15-24, 25-34 weeks of gestation) in 1,366 nulliparous women. After delivery, women were classified into three categories: normal, pre-eclampsia, transient hypertension. The predictive performance of the tests, either used alone or in combination (stepwise multiple logistic regression), was assessed and compared to that of the mean arterial pressure. Pre-eelampsia developed in 109 of the pregnant women. Significant difference in the mean value of the tests between the normal and pre-eclamptic women, after Bonferroni correction, was only observed for three biochemical variables: progesterone, mean corpuscular volume and haptoglobin. Multiple logistic regression models were developed using the biochemical and clinical variables. Their predictive performance was compared with that of the mean arterial pressure using a cut-off value at a given specificity of 80%. The sensitivity, positive and negative predictive values for mean arterial pressure were respectively 46.6%, 23.5%, 92.0% at the second visit and 52.5%, 26.0% and 92.6% at the third visit. A logistic model for the second visit that included the mean arterial pressureshowed a sensitivity of 57.1%, a positive predictive value of 26.9% and a negative predictive value of 93.7%. For the model developed for the third visit, the corresponding values were 66.1%, 30.0% and 94.6%. Combination of the tests in a stepwise multiple regression model offered little advantages over the single use of mean arterial pressure.
CLINICALBIOCHEMISTRY,VOLUME 26, APRIL 1993
-ffq
A preliminary prospective evaluation of
b i o c h e m i c a l m a r k e r s f o r fetal t r i s o m i e s Forest, J-C., Mass6, J., Rousseau, F., Moutquin, J.M., Brideau, N.A., B61anger, M. and Bastide, A. D6partements de Biochimie et Obst~trique-Gyn6cologie, Facult~ de M6Aecine, Universit6 Laval, Cit6 Universitaire, Qu6bec, G1K 7P4, Canada We are currently measuring three markers (alphafetoprotein, estriol, choriogonadotropin) in pregnant women of all ages to determine their value in screening for fetal trisomies. Our study population consists of 9,000 women who attended the perinatal clinics of six hospitals and had signed a consent form. A blood specimen was obtained between 10 and 19 weeks gestation and the serum was stored at -20°C. Alphafetoprotein and choriogonadotropin were rrleasured with Enzymun-Test R enzymeimmunoassays (Boehringer Mannheim Canada). Unconjugated estriol was assayed with an ultrasensitive radioimmunometric assay (DSL Canada). Random samples from unaffected pregnancies were used to determine the distribution of results at each week. The maternal agespecific risk (a priori odds) was determined using published results (Br J Obstet G~aecol 1987; 94:387-402). The woman's multiples of median for each marker were used to calculate a likelihood ratio using the distribution parameters derived from our population. The posteriori odds were obtained by multiplying the a priori odds by the likelihood ratios. The results were considered positive if the calculated risk was equal to or higher than that of women of 35 years. Thirteen cases of singleton trisomy 21 occurred. Using age in combination with the three biochemical markers, 53.8% of these cases have been detected. No cases that happened in women 35 years or older would have been missed. These results confirm the sensitivity published by other groups. The application of this screening strategy in the older women does not appear to affect detection of a trisomic foetus while obviating a number of unnecessary amniocenteses. II
METHOD AND INSTRUMENT EVALUATION
[]
A t i m e - s t u d y a n a l y s i s to e v a l u a t e a l b u m i n c a r b a m y l a t i o n in vitro Balion, C.M., Caines, P.S.M., Draisey, T.F. and Thibert, g.J. Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, N9B 3P4, Canada
135
37TH ANNUAL CONFERENCE OF THE CANADIAN SOCIETY OF CLINICAL CHEMISTS
Carbamylation is a rapid, nonenzymatic reaction occurring between isocyanic acid, the spontaneous dissociation product of urea, and proteins. This process is increased in vivo in chronic renal failure patients, who have high urea levels. Human albumin (40 mg/mL) was incubated at room temperature with 0.1M sodium cyanate in 0.1M sodium phosphate buffer, pH 7.4, for four days. During the incubation, aliquots were removed and the albumin separated from excess cyanate by means of a P-6DG exclusion column. The desalted albumin samples were applied to an ultrathin polyacrylamide gel (Bio-Lyte 4/6) and focused using a Bio-Rad ° Mini IEF Cell. The degree of carbamylation was measured colorimetrically using diacetyl monoxime and expressed as absorbance units/mg albumin. The pI of the carbamylated albumin decreased from 4.9 (native albumin) to 4,2 after 40 h of incubation. Similarily, the number of carbamyl groups increased in proportion to the change in pI. These results indicate that the carbamylation process can be easily detected in vitro and that a saturating level of carbamylation occurs. The techniques employed in this study may be useful in evaluating the extent of carbamylation in patients with chronic renal failure.
D
Detection of PCR-amplified chronic myelogenous l e u k e m i a - s p e c i f i c m R N A sequences by i m m u n o f l u o r o m e t r y
Bortolin, S. and Christopoulos, T.K. Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Street, Windsor, Ontario, N9B 3P4, Canada The Philadelphia chromosome (Ph) is present in >95% of chronic myelogenous leukemia (CML) patients. It involves a reciprocal translocation, which fuses the ABL protooncogene to the BCR gene. Expression of the fusion gene results in a leukemia-specific BCR-ABL mRNA. To facilitate the diagnosis and monitoring of CML, we developed a highly sensitive, nonisotopic microtiter platebased assay for leukemia-specific mRNA in leukocytes. K562, a Ph-positive cell line is used as a positive control. mRNA is isolated from 103-106 leukocytes. A cDNA copy is synthesized by reverse transcription. Then, the diagnostically useful cDNA sequence is amplified by nested polymerase chain reaction (PCR). PCR I uses unlabeUed 21mer and 24met primers homologous to BCR and ABL, respectively. PCR II employs 22mer primers, 5'end-labelled with digoxigenin (DIG) and biotin. PCR I is performed for 25 cycles followed by 15 cycles of PCR II. The 200 bp amplified product carries biotin at one end and DIG at the
136
other. The reaction mixture is transferred to streptavidincoated microtiter wells where the PCR product is captured. Afterwards, alkaline phosphatase-labelled antidigoxigenin antibody is added. Fluorosalicylphosphate is used as substrate. The released fluorosalicylate, forms highly fluorescent complexes with Tb3+-EDTA. The fluorescence is measured with a time-resolved fluorometer. Amplified product corresponding to a single leukemic cell can be detected. Only Ph-positive cells give a double-labelled product. The whole procedure takes about 6 h. The proposed method avoids time-consuming steps such as electrophoresis and Southern blot and the use of radioisotopes.
Fq
Quantitative polymerase chain reaction analysis of pathogenic DNA sequence using an internal DNA sequence standard constructed by recombinant DNA methodology
Chan, A., Krajden, M. and Diamandis, E.P. Department of Clinical Biochemistry and Microbiology, The Toronto Hospital, 200 Elizabeth Street, Toronto, Ontario, M5G 2CA, Canada The successful development of sensitive and reliable quantitative assays for polymerase chain reaction (PCR) products using High Performance Liquid Chromatography (HPLC) have opened up new research and clinical applications that promote the understanding of pathology, pharmaco-toxicology, therapeutic management and diagnosis ofvarions diseases. However, it has been difficult to correlate the amount of PCR products output to the amount of DNA targets input which should be the more meaningful data for clinical applications. There are multiple factors leading to this analytical problem, such as the reproducibility of PCR thermoeycling runs, the integrity of reagents used, the PCR conditions and kinetics, the sample preparation methodologies and matrix interference, etc. By constructing a recombinant cytomegalovirus (CMV) control target plasmid which contained a modified CMV AD169 sequence that shared the PCR primer sequence of native CMV AD169 and co-amplified both the unknown and control plasmid targets, we were able to by-pass some of the kinetic PCR problems and to quantify native CMV AD169 targets. PCR co-amplification of the CMV control and CMV native plasmid targets resulted in two distinctively separable PCR products of 362 bp and 152 bp respectively, with a 15 rain HPLC run. During PCR coamplification there was an overall decrease as well as competition in the synthesis of the PCR products from both plasmid targets as compared to non-co-amplified
CLINICAL BIOCHEMISTRY, VOLUME 26, APRIL 1993