A capillary zone electrophoresis method for determining N-acetylneuraminic acid in glycoproteins and blood sera.

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per-O-benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS<1.98%) and a linearity range from 5 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.

Identification, quantification and fine structural characterization of glycosaminoglycans from uterine leiomyoma and normal myometrium.

The type, amount and fine chemical composition of glycosaminoglycans (GAGs) present both in human normal myometrium and uterine leiomyoma have been studied. GAGs were fractionated by ion-exchange chromatography on DEAE-Sephacel, isolated by gel-permeation chromatography on Sepharose CL-6B and characterized using electrophoresis in cellulose acetate membranes, specific enzymic treatments and analysis by high-performance capillary electrophoresis (HPCE). No statistical intrabatch differences in total GAG content in both tissues were identified, whereas significant interbatch differences between normal myometrium and uterine leiomyoma were recorded. Hyaluronan (HA), chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate (HS) and keratan sulphate (KS) were identified in both tissues. Statistically significant (P

Importance of high-performance liquid chromatographic analysis of serum N-acylneuraminic acids in evaluating surgical treatment in patients with early endometrial cancer.

The objectives of this study were the quantification of the two major sialic acid (Sia) forms - N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acids (Neu5Gc) - in serum before and after surgical treatment of early endometrial cancer and the relation of their levels with the progress of surgical therapy. The major Sia forms were liberated from sera glycoconjugates by mild acid hydrolysis, separated as per-O-benzoylated derivatives by a highly sensitive reversed-phase HPLC method and detected at 231 nm. Total Sia content in sera of healthy women was not related to age and body weight. Neu5Ac was identified as the major Sia in sera from both cancer patients, healthy individuals as well as in tissue specimens (> or = 94% of total Sia). In patients with endometrial cancer the total Sia level before surgical treatment (709.5 +/- 306.5 mg/l) was significantly higher (p < or = 0.0001) than that of the control group (213.5 +/- 88.7 mg/l). The elevation in Sia level was exclusively due to Neu5Ac. Following surgical therapy, serum Neu5Ac levels (699.4 +/- 305.6 mg/l) were significantly decreased (305.9 +/- 114.5 mg/l). In one case, where Neu5Ac level was increased 15 days and eight months after surgery (1.8 and 2.5 times as compared to control, respectively), a metastasis not detected during surgery was recorded. The obtained results suggest that Neu5Ac level in serum may be used as a tumor marker in evaluating the suitability of surgical treatment in early endometrial cancer.

Effect of antenatal administration of thyrotrophin releasing hormone on fetal flow velocity waveforms.

AIM: To determine whether antenatal administration of thyrotrophin releasing hormone (TRH), to promote lung maturation, alters blood flow through the fetal middle cerebral, umbilical artery, or ductus arteriosus and through the maternal uterine arteries. METHODS: The effect of transplacentally administered TRH on the fetal circulation was prospectively evaluated in 30 patients between 24 and 34 weeks' gestation. TRH (400 micrograms) was given to the mother intravenously either as a bolus or an infusion. Fetal effects were determined by measuring the maximum velocity and pulsatility index (PI) in middle cerebral artery, ductus arteriosus, uterine artery and umbilical artery Doppler waveforms. Measurements were made immediately before, and 10 and 60 minutes after maternal TRH administration. RESULTS: Intravenous injection of TRH had no significant effect on PI in the uterine, umbilical, or middle cerebral artery and the ductus arteriosus within 60 minutes of administration in either group. CONCLUSION: The antenatal use of TRH in conjunction with steroids for fetal lung maturity does not affect utero-placental or fetal haemodynamic variables, as measured by Doppler. These findings, therefore, do not support the suggestion that antenatal intravenous administration of TRH either as bolus or infusion may have immediate adverse vascular effects in the fetus.

Echogenic material in the fetal gallbladder in a surviving monochorionic twin.

Fetal gallstones or echogenic sludge in the fetal gallbladder have rarely been reported prenatally despite the increasing number of ultrasound scans performed during pregnancy. In this report we present a case in which diffuse echogenic material was detected in the fetal gallbladder in a surviving monochorionic twin. This report identifies another predisposing factor for fetal gallstones/sludge in the perinatal period.

Accuracy of prenatal diagnosis of renal agenesis with color flow imaging in severe second-trimester oligohydramnios.

OBJECTIVE: Our purpose was to examine the potential of color flow imaging to assess the presence of renal arteries in second-trimester pregnancies complicated by severe oligohydramnios. STUDY DESIGN: Thirty-three consecutive second-trimester pregnancies referred with severe oligohydramnios were prospectively studied with high-resolution color Doppler ultrasonography to establish the presence or absence of renal arteries. Prenatal findings were correlated with the presence or absence of fetal kidneys at postmortem or postnatal examination. RESULTS: Neither renal artery was visualized in eight fetuses; postmortem examination confirmed bilateral renal agenesis in seven and unilateral renal agenesis with a contralateral atrophic multicystic kidney in the other. Only one renal artery was seen in three; postmortem examination demonstrated unilateral renal agenesis in two fetuses and bilateral multicystic dysplastic kidneys in the other. Postmortem or postnatal evaluation confirmed the presence of both kidneys in all 22 fetuses in which both renal arteries were identified prenatally. CONCLUSIONS: Color Doppler ultrasonography is useful in the prenatal evaluation of fetuses with severe second-trimester oligohydramnios to demonstrate the presence or absence of renal arteries. This technique should be added to the armamentarium of prenatal tests to evaluate second-trimester fetuses with severe oligohydramnios.

Ultrasonographic measurement of the dividing membrane in twin pregnancy during the second and third trimesters: a reproducibility study.

OBJECTIVE: Our purpose was to examine the reproducibility of intertwin membrane thickness measurements used to predict chorionicity in twin pregnancies. STUDY DESIGN: Twenty-seven twin pregnancies were scanned with high-resolution ultrasonography on 52 occasions during the second and third trimesters. Two observers, blind to other criteria of chorionicity, measured the dividing membrane twice in five different sites (total measurements 1040). The data were log-transformed and the coefficient of repeatability calculated as a measure of intraobserver variability. Interobserver variability was estimated by the Bland and Altman 95% limits of agreement. Random variation was assessed with the restricted maximum likelihood procedure in Genstat. RESULTS: The overall estimate of the coefficient of repeatability was 2.14, indicating that 95% of repeated measurements would be expected to be within 114% of each other. Measurements taken close to the placenta (up to 3 cm) were the most repeatable and displayed no bias when repeated. Coefficients of repeatability at this site ranged from 1.42 to 1.91, with no evidence of consistent differences between monochorionic and dichorionic twins. Gestational age was not significantly associated with membrane thickness for any of the models. The pregnancy type x subject x observer and pregnancy type x subject x site interactions were statistically significant (p < 0.001 and p < 0.005, respectively), implying that interobserver variability depends on the subject being measured, the site of sampling, and chorionicity. CONCLUSION: Ultrasonographic measurement of membrane thickness has high intraobserver and interobserver variability in the second and third trimesters. Our findings provide an explanation for the suboptimal accuracy reported with this method in determining chorionicity in the second and third trimesters.

Early prenatal diagnosis of holoprosencephaly: the value of transvaginal ultrasonography.

Holoprosencephaly is a rare condition characterized by different degrees of fused ventricles resulting from the failure of the prosencephalon to cleave during embryonic life. In this report we present a case of alobar holoprosencephaly diagnosed prenatally at 15 weeks' gestation in a woman with a family history of open neural tube defect. The diagnosis was initially missed because only suboptimal views of the fetal brain were obtained with transabdominal ultrasonography due to fetal position; however, a better depiction of brain anatomy was obtained using the transvaginal approach, allowing the detection of a single ventricle and fused thalami. We conclude that transvaginal ultrasonography is not only important for the early prenatal diagnosis of central nervous system abnormalities but also for enhancing brain visualization when transabdominal examination of the fetal head is limited due to maternal and fetal factors.

Prenatal findings in generalized amyoplasia.

Amyoplasia is a rare, sporadic condition characterized by different degrees of maldevelopment of the skeletal muscles, which are replaced by fibrous and fatty tissue. In this report, we present a case of generalized amyoplasia presenting at 19 weeks' gestation. The most striking finding was the absence of fetal movements, resulting in severe multiple congenital contractures, hydrops, and polyhydramnios. At autopsy, histological examination of the skeletal muscle showed small groups of poorly developed fibres within areas of fat. This report suggests that generalized amyoplasia could be a common cause of severe forms of multiple congenital contractures, but is probably underdiagnosed at post-mortem because of inadequate examination of muscles. Definitive diagnosis is important in determining the risks of recurrence in these cases.

Exencephaly in autosomal dominant brachydactyly syndrome.

Exencephaly was diagnosed at 17 weeks in a 27-year-old primigravida with abnormalities of the hands and a family history suggestive of autosomal dominant brachydactyly and clinodactyly. In this family there was also a history of 'anencephaly'. To our knowledge, this is the first report on the association of exencephaly and autosomal dominant brachydactyly. As the relationship between hand and cranial anomalies is well established, we suggest that this association in our case could be due to a defect in the same gene.

Fetal blood mononuclear cell division in normal and pathological pregnancies.

Fetal blood mononuclear cell division was measured using flow cytometry in 53 normal pregnancies and 51 pathological pregnancies complicated either by anaemia due to red blood cell isoimmunisation (RCI: n = 21), intrauterine growth retardation (SGA: n = 13) or abnormal karyotype (n = 17). In normal pregnancy, mononuclear cell division rates decreased with gestational age from a mean of 1.8% at 18 weeks to 1% at 40 weeks. Furthermore, there was a significant association between cell division and erythroblast count. The rates of cell division and erythroblast count were significantly increased in the chromosomally abnormal fetuses, and significantly decreased in the transfused RCI fetuses compared to the controls. Although the erythroblast count was elevated in the SGA fetuses, the mononuclear cell division was not significantly different from the controls. Fetal blood mononuclear cell division is elevated in early pregnancy and in chromosomally abnormal fetuses, probably as a consequence of increased numbers of circulating haemopoietic precursors. Mononuclear cell division is decreased in transfused RCI fetuses as a consequence of suppressed erythropoiesis. In SGA fetuses, despite the increased erythropoietic stimulation and erythroblastosis, cell division is not increased.

Response of tertiary centres to pressure changes. Is there a mechano-electrical association?

STUDY OBJECTIVE: To investigate the mechanism of pressure related ventricular arrhythmias by examining them during atrioventricular (AV) block. DESIGN: Complete AV block, where all ventricular beats are ectopic, was induced by AV node ablation and/or by toxic digitalisation, and rhythm changes were studied while arterial blood pressure was repeatedly raised and lowered. SUBJECTS: 15 anaesthetised mongrel dogs, weight 15-28 kg, were used. AV block was induced in eight by chemical or mechanical ablation of the AV node. In five of these and in seven other dogs, 5.0-7.5 mg digoxin was also given. MEASUREMENTS AND RESULTS: Following AV block due to ablation, a heart rate increase (or no change) was found in 87.5% of 56 arterial pressure increases produced by elevation of an open arterial blood reservoir or by metaraminol infusion, but in only 21.8% of 55 pressure decreases caused by arterial bleeding (p much less than 0.001). Following AV block due to digitalisation, the equivalent figures were 96% of 50 pressure increases and 27.3% of 55 pressure decreases (p much less than 0.001). While arterial pressure was increased there was moderate acceleration of the escape rhythm, then appearance of premature ventricular beats, then non-sustained and finally sustained ventricular tachycardia. The reverse occurred, with some hysteresis, on decreasing the arterial pressure. In five of the digitalised animals, arterial pressure reduction to nearly zero caused reproducible sudden arrest, with resumption of the ordinary escape rhythm on increasing the pressure again. CONCLUSIONS: The findings suggest the possibility of two kinds of ectopic rhythm in AV block: the "normal" escape rhythm which is only moderately affected by arterial pressure changes; and an "abnormal" faster pressure dependent rhythm which is generated by high arterial pressure and abolished by pressure near zero, as if there were a mechano-electrical association. This abnormal rhythm may prevail completely in digitalis toxicity so that if cardiac arrest occurs, no automaticity can be expected to appear unless arterial pressure is raised.