Developmental aspects of pteridine metabolism and relationships with phenylalanine metabolism

Large variations of pteridine elimination occur in childhood, due to the ontogenic development of the metabolism of tetrahydrobiopterin. The main feature is the slow maturation of biopterin synthesis whereas neopterin synthesis is high at birth; thus a high neopterin to biopterin ratio (4.4 ± 2.1) occurs in the neonatal period, a ratio which then decreases to adult values (0.5 ± 0.2). Comparing pteridine elimination of PKU patients with that of controls of the same age, a high excretion of biopterin and, to a lesser extent, of neopterin is found. In normal subjects, following an oral phenylalanine load, biopterin levels in urine and serum also increase, whereas variations of neopterin concentration are small. In rats, phenylalanine also leads to an increase of serum biopterin whereas liver biopterin decreases. This suggests that the main explanation for the biopterin increase in serum and in urine by phenyl-alanine is a release of the intracellular biopterin by the aminoacid.     

Altered urinary excretion of pteridines in neoplastic disease. Determination of biopterin,neopterin, xanthopterin,and pterin

Urinary pteridine concentrations in healthy control subjects and patients with cancer and non-malignant diseases were determined by HPLC and TLC after partial purification by ion exchange and Sephadex chromatography. Elevated concentrations of neopterin were found in 70% of the 50 cancer patients investigated. In patients with non-Hodgkin's lymphoma (seven cases) or with liver metastases (12 cases) neopterin concentrations were significantly higher than in control subjects (p < 0.01). Biopterin was less frequently increased (22%). Xanthopterin was generally raised when neopterin and/or biopterin excretion was high. Neopterin/biopterin ratios were higher in some patients with cancer or with severe renal insufficiency than in controls. These findings suggest that alterations in pteridine metabolism are common in malignant disease. The pathogenic, diagnostic and therapeutic significance of these changes remains to be established.     

Highly sensitive, specific enzyme-linked immunosorbent assay of neopterin and biopterin in biological samples.

An enzyme immunosorbent assay of neopterin and biopterin on a polystyrene microtiter plate has been developed. A conjugate of neopterin or biopterin to bovine serum albumin was used to raise a specific antiserum against neopterin or biopterin in rabbits. An incubation mixture of the antiserum and samples prepared from human serum underwent another antigen-antibody reaction with the hapten fixed on the microtiter plate. The amount of antibody bound to the fixed hapten, which is inverse to the amount of hapten in the sample, was determined by using anti-rabbit IgG-horseradish peroxidase conjugate in a usual manner by measuring absorbance at 490 nm after reaction with o-phenylenediamine and hydrogen peroxide. The minimal detectable amounts of neopterin and biopterin were approximately 0.1 pmol. The specificity of the assay was so high that the assay system for neopterin completely distinguished it from biopterin, as judged from the cross-reaction of 0.002%, and vice versa. The amounts of neopterin and biopterin in human serum determined by the present method agreed well with those determined by high-performance liquid chromatography. We used the present method to determine the concentrations of neopterin in serum from healthy control subjects and patients with cancers and systemic lupus erythematosus; the results were consistent with literature data.     

Polarization fluoroimmunoassay of biopterin and neopterin in human urine

A polarization fluoroimmunoassay has been developed for the routine determination of biopterin and neopterin levels in human urine. The method employs fluorescein-labeled biopterin and neopterin. The assay is fast (incubation time: 2 min) and no separation step is required prior to measurement of fluorescence polarization. Linearity, recovery and precision were satisfactory. Estimations of biopterin and neopterin levels in human urine samples closely correlated with those obtained by radioimmunoassay.     

Liquid-chromatographic measurement of biopterin and neopterin in serum and urine

We report an improved "high-performance" liquid-chromatographic (HPLC) method for measuring biopterin and neopterin in serum and urine. Specimens are acidified, treated with iodine in 0.2 mol/L trichloroacetic acid, party purified on Bio-Rad MP-50 cation-exchange columns, and analyzed by reversed-phase HPLC with fluorometric detection. The minimal concentration of biopterin detectable is 0.3 micrograms/L in a 50-microL injection. The total CV is less than or equal to 10%. Improvements over other reported methods include the use of a single, simplified sample-preparation step with a Baker-10 SPE System, and a guard column to increase analytical column stability and analyte recovery. The assay is semiautomated to reduce technician time and improve precision. Mean observed values for biopterin and neopterin in sera of normal human adults were 1.64 and 5.52 micrograms/L, respectively. The mean ratio of neopterin to biopterin in acidified adult urine samples was lower than that found in matched nonacidified samples (n = 10). Serum specimens from diagnosed phenylketonuric (PKU) and hyperphenylalaninemic patients were also analyzed for biopterin and neopterin; the findings agreed with reported values for similar patients. One patient, previously identified as an atypical PKU patient, showed serum values of neopterin and biopterin suggestive of a defect in biopterin synthesis.     

An improved determination of total glycosaminoglycans in body fluids by formation of complexes with quinacrine: changes in amniotic fluid total glycosaminoglycans during normal pregnancies and in pregnancies at risk for mucopolysaccharidoses.

Acidic glycosaminoglycans form insoluble complexes with quinacrine and this has been exploited for their analysis in blood, urine and amniotic fluid. The method is specific for glycosaminoglycans including keratan sulphate and the samples do not have to be deproteinized. Values for normal urine, serum and amniotic fluid are presented. Urinary total glycosaminoglycans excreted by patients with mucopolysaccharidoses were also determined. The normal changes in amniotic fluid total glycosaminoglycans have been measured between 14 weeks' gestation and term, and values are given for amniotic fluid total glycosaminoglycans in several pregnancies at risk for mucopolysaccharidoses. It is suggested that this method is a potentially valuable analytical tool in the pre-natal diagnosis of mucopolysaccharidoses.     

Bile acid pattern in human amniotic fluid

Individual bile acids were determined in twenty-nine amniotic fluid specimens obtained from twenty-six women between the 32nd and 41st week of gestation. Total bile acid concentration ranged from 0.4 to 4.8 mumol/l with a mean of 1.57 mumol/l. Besides the two major bile acids of man, cholic acid and chenodeoxycholic acid, 3beta-hydroxy-5-cholenoic acid was found in all, lithocholic acid in ten and deoxycholic acid in nine of the twenty-nine amniotic fluid samples. 3beta-Hydroxy-5-cholenoic acid averaged 39.8% of total bile acids during 32-37 weeks of gestation and 20.2% at term (P less than 0.01). These findings point towards important differences between fetal and adult bile metabolism and may reflect maturation of hepatic bile acid biosynthesis near term.     

The concentration of glucose in the amniotic fluid in a high risk pregnancy group]

Amniotic fluid glucose levels were measured in 125 pregnant women included in high-risk group in respect of perinatal pathology. In the reference group the measurements have demonstrated an agreement between glucose concentrations in the amniotic fluid and gestation term and an inverse relationship between pregnancy progress and glucose level, whereas in diabetics this relationship was direct. Fetal developmental defects were associated with elevated glucose content in the amniotic fluid, concomitant placental abnormalities were conducive to reduction of these levels, and no correlations between this parameter and pregnancy terms were observed.     

Maternal Intravenous Administration of Azithromycin Results in Significant Fetal Uptake in a Sheep Model of Second Trimester Pregnancy

Treatment of intrauterine infection is likely key to preventing a significant proportion of preterm deliveries before 32 weeks of gestation. Azithromycin (AZ) may be an effective antimicrobial in pregnancy; however, few gestation age-approriate data are available to inform the design of AZ-based treatment regimens in early pregnancy. We aimed to determine whether a single intra-amniotic AZ dose or repeated maternal intravenous (i.v.) AZ doses would safely yield therapeutic levels of AZ in an 80-day-gestation (term is 150 days) ovine fetus. Fifty sheep carrying single pregnancies at 80 days gestation were randomized to receive either: (i) a single intra-amniotic AZ administration or (ii) maternal intravenous AZ administration every 12 h. Amniotic fluid, maternal plasma, and fetal AZ concentrations were determined over a 5-day treatment regimen. Markers of liver injury and amniotic fluid inflammation were measured to assess fetal injury in response to drug exposure. A single intra-amniotic administration yielded significant AZ accumulation in the amniotic fluid and fetal lung. In contrast, repeated maternal intravenous administrations achieved high levels of AZ accumulation in the fetal lung and liver and a statistically significant increase in the fetal plasma drug concentration at 120 h. There was no evidence of fetal injury in response to drug exposure. These data suggest that (i) repeated maternal i.v. AZ dosing yields substantial fetal tissue uptake, although fetal plasma drug levels remain low; (ii) transfer of AZ from the amniotic fluid is less than transplacental transfer; and (iii) exposure to high concentrations of AZ did not elicit overt changes in fetal white blood cell counts, amniotic fluid monocyte chemoattractant protein 1 concentrations, or hepatotoxicity, all consistent with an absence of fetal injury.     

Is there a relationship between fetal weight and amniotic fluid index?

OBJECTIVE: To establish whether there is a relationship between the amniotic fluid index and estimated fetal weight in the third trimester. The presence of a relationship would require adjustment of amniotic fluid index to take account of estimated fetal weight with potential improvement in its prediction of adverse perinatal outcomes. METHODS: Paired measurements of amniotic fluid index and estimated fetal weight from 274 low-risk pregnancies enrolled in a longitudinal study of fetal growth. Measurements were made at fortnightly intervals from 30 weeks' gestation until delivery. A relationship between amniotic fluid index and estimated fetal weight was sought at gestational age week intervals of 30-32, 33-35, 36-38 and 39-41. RESULTS: One thousand and three pairs of measurements of amniotic fluid index and estimated fetal weight were available for analysis. Mean amniotic fluid index decreased towards term as expected. There was no correlation between amniotic fluid index and estimated fetal weight. Furthermore, there was no correlation between amniotic fluid index and estimated fetal weight at any of the gestational age intervals. CONCLUSIONS: There is no clinically relevant correlation between amniotic fluid index and estimated fetal weight. It should remain clinical practice to take account of gestational age when interpreting amniotic fluid index but it is not necessary to make adjustments for estimated fetal weight.     

Accuracy of ultrasonography in evaluating amniotic fluid volume at less than 24 weeks' gestation.

The purpose of this investigation was to evaluate the accuracy of common sonographic techniques in assessing the amniotic fluid volume in pregnancies of less than 24 weeks' gestation. Patients at less than 24 weeks' gestation undergoing an amniocentesis for the placement of prostaglandin F2 alpha for termination (because of genetic or fetal anomalies, or both) were assessed for amniotic fluid volume. All fetuses were alive at the time of prostaglandin instillation. The amniotic fluid index and two-diameter pocket were used to determine the amniotic fluid volume. Prior to the prostaglandin instillation, the amniotic fluid volume was determined with para-aminohippurate using a diazo dye reaction with spectrophotometric analysis. The amniotic fluid volume was determined in 21 pregnancies between 15 and 24 weeks' gestation, yielding volumes ranging from 189 to 1840 ml. Using published standards for amniotic fluid volume in singleton pregnancies, oligohydramnios was present in three gestations, the volume was found to be normal in 15, and hydramnios complicated three pregnancies. The two-diameter pocket identified the amniotic fluid volumes correctly more often (18 of 21 [85.7%]) than the amniotic fluid index (10 of 21 [47.6%]) (P = 0.02). Normal amniotic fluid volume was identified in nine of 15 (60%) pregnancies by the amniotic fluid index and in 14 of 15 (93.3%) by the two-diameter pocket (P = not significant). Abnormal amniotic fluid volumes, oligohydramnios, and hydramnios were recognized more often by the two-diameter pocket (66.7%) than by the amniotic fluid index (1 of 6 [16.7%], P = not significant).     

Assessment of homocysteine, neopterin and nitric oxide levels in Beh?et's disease.

BACKGROUND: Beh?et's disease is a multisystemic immunoinflammatory disease with a wide variety of clinical manifestations, whereas recurrent aphthous stomatitis is a local oral disease. The aim of this study was to examine the distribution of homocysteine levels in patients with active Beh?et's disease, possible association of homocysteine with nitric oxide and neopterin levels, and to characterize the differences between patients with Beh?et's disease and those with recurrent aphthous stomatitis in terms of these parameters compared with healthy controls. METHODS: A total of 23 patients with active Beh?et's disease, 25 patients with recurrent aphthous stomatitis as positive controls, and 21 healthy subjects were included in this study. Serum homocysteine and neopterin levels were measured flourimetrically by HPLC. Serum nitric oxide production was assayed by measuring total nitrite levels with Griess reagent. RESULTS: Significantly higher homocysteine (12.9+/-3.3 micromol/L) and lower nitric oxide (41.5+/-10.9 micromol/L) and neopterin (6.4+/-1.0 nmol/L) levels were observed in patients with Beh?et's disease compared with healthy controls (10.7+/-2.0 micromol/L, 49.7+/-16.2 micromol/L, 8.7+/-2.2 nmol/L, respectively) (p<0.03 for neopterin, p<0.04 for homocysteine and nitric oxide). However, homocysteine, nitric oxide, biopterin and neopterin levels and the neopterin/biopterin ratio for recurrent aphthous stomatitis patients were not significantly different compared to healthy controls. A significant positive correlation was observed between serum homocysteine and serum neopterin/biopterin ratio in patients with Beh?et's disease (r=0.975, p<0.005). CONCLUSIONS: In contrast to recurrent aphthous stomatitis, there is a higher prevalence of hyperhomocysteinemia in Behcet's disease. Homocysteine may have deleterious effects on the pathology of Behcet's disease by decreasing nitric oxide levels and interfering with the immune system.     

Early amniocentesis and amniotic fluid organic acid levels in the prenatal diagnosis of organic acidemias

Organic acids were studied in amniotic fluids taken by early amniocentesis between 6 to 12 weeks in control pregnancies. The prenatal diagnosis of methylmalonic acidemia and propionic acidemia could be made by the measurement of methylmalonic and methylcitric acid respectively in the amniotic fluid taken simultaneously with chorionic villi at the 11th week of gestation.     

Is oligohydramnios a risk factor for infection in term premature rupture of membranes?

Our objective was to determine if a reduced volume of amniotic fluid is a risk factor for microbial invasion of the amniotic cavity in women with rupture of membranes at term. Transabdominal amniocentesis under ultrasound guidance was used to evaluate the microbiological state of the amniotic cavity in 53 patients with term premature rupture of membranes before vaginal examination. Amniotic fluid index was measured prior to the procedure in all cases. The prevalence of microbial invasion of the amniotic cavity was 32.1% (17/53). Women with microbial invasion of the amniotic cavity had a significantly lower median amniotic fluid index than did women without evidence of infection (median 4.4 cm, range 1.0-8.1 vs. median 7.8 cm, range 1.3-14.4, respectively; p < 0.001). An amniotic fluid index of < 5 cm had a sensitivity of 71% (12/17) a specificity of 89% (32/36), a positive predictive value of 75% (12/16) and a negative predictive value of 87% (32/37) in the prediction of microbial invasion of the amniotic cavity. Among women who delivered vaginally, those with an amniotic fluid index of < 5 cm had a higher rate of endometritis than those with an amniotic fluid index of >or= 5 cm (19% (3/16) vs. 0% (0/26), respectively; p < 0.05). We conclude that women with term premature rupture of membranes and an amniotic fluid index of < 5 cm are at an increased risk for microbial invasion of the amniotic cavity and puerperal infection after a vaginal delivery.     

足月妊娠羊水超声组织定征及临床应用价值研究

目的：对足月孕妇羊水进行超声回声强度（EI）定量分析，为临床及时诊断胎儿宫内窘迫提供客观的羊水性状指标。方法：对27例足月妊娠孕妇羊水池进行EI定量测定。根据手术分娩后羊水性状分为正常羊水组（16例）及羊水粪染组（11例），比较两组间EI灰阶（GS）、分贝（dB）差异。结果：羊水粪染组灰阶及分贝最大值明显高于正常羊水组。结论：超声组织定征可用于临床前羊水性状的监测。     

N-Acetyl-beta-hexosaminidase isoenzymes of amniotic fluid and maternal serum. Their relevance to prenatal diagnosis of the GM2 gangliosidoses.

The isoenzymes of N-acetyl-beta-hexosaminidase were quantitated in 30 amniotic fluid and 13 maternal serum samples collected between 11 and 40 weeks of gestation using DEAE-Sephadex A-25 chromatography. Isoenzymes A and B consitituted the major components of most amniotic fluids but seven samples characterized by high N-acetyl-beta-hexosaminidase activities contained high proportion of an isoenzyme apparently identical to isoenzyme P of maternal serum. This passage of maternal serum N-acetyl-beta-hexosaminidase into the amniotic cavity could lead to false negative diagnosis of type B and type O GM2 gangliosidoses if the diagnosis rely solely upon isoenzyme analysis in amniotic fluid. However, the release of maternal enzyme into amniotic fluid seems to be restricted to the third trimester of gestation and should not interfere with prenatal diagnosis of the GM2 gangliosidoses ususally performed at an earlier stage of gestation.     

Oligohydramnios associated with bilateral multicystic dysplastic kidneys: prenatal diagnosis at 15 weeks' gestation

Lethal fetal renal malformations are invariably associated with oligohydramnios in late antennatal ultrasound examinations. Controversy exists concerning the contribution of fetal urine production to amniotic fluid volume prior to 20 weeks' gestation. We report a pregnancy complicated by maternal gold usage and fetal bilateral multicystic dysplastic kidney disease in which oligohydramnios was detected at 12 weeks' gestation. To our knowledge, no previous cases of lethal fetal renal anomalies have been made in the first trimester. The early diagnosis of fetal renal anomalies, fetal renal contribution to amniotic fluid production, and possible teratogenicity of gold therapy is discussed.     

Cholesterol in amniotic fluid, determined by gas chromatography.

Cholesterol was extracted from amniotic fluid, saponified, converted to its trimethylsilyl derivative, and gas chromatographed, with cholesteryl acetate as the internal standard. The method is sufficiently accurate and precise for use with the range of concentrations of cholesterol found in amniotic fluid (5 to 48 mg/litre). Total cholesterol was measured in amniotic fluids collected at different stages of gestation. No significant trend or change was observed nor was cholesterol in the amniotic fluid and the mother's serum correlated at any stage of gestation. Thus we conclude that cholesterol is not a useful indicator of fetal age or maturity. Cholesterol concentrations in amniotic fluid from complicated pregnancies were within the range found for normal pregnancies.     

Ultrasonographic demonstration of floating particles in amniotic fluid

This prospective study of 131 pregnant women was designed to determine the incidence and significance of floating particles in amniotic fluid. Floating particles ranging in size from 1 to 5 mm were seen in the amniotic fluid of pregnant women at 15 to 40 weeks' gestation. Since vernix is rarely present before 35 weeks' gestation, a source other than flakes of vernix must be sought to explain the floating particles in amniotic fluid in early gestations. There was no significant difference in the sizes or numbers of particles at different gestational ages (from 15 to 40 weeks). Therefore, it is concluded that ultrasonographic demonstration of floating particles in amniotic fluid cannot be considered an indicator of fetal maturity.     

妊娠晚期羊水过少112例临床分析

目的:探讨羊水过少的相关因素及围产儿的预后。方法:采用回顾性分析方法,对妊娠晚期羊水过少112例与羊水正常100例进行对比分析。结果:羊水过少多发生在孕40周后,与胎儿宫内发育迟缓(IUGR)和延期、过期妊娠有关;羊水过少组围产儿不良预后明显高于羊水正常组(P<0.01)。结论:妊娠晚期羊水过少是胎儿宫内慢性缺氧的标志,对围产儿预后有严重影响,应加强产前监测。剖宫产是处理妊娠晚期羊水过少及降低围产儿死亡率的重要措施。