Microdetermination of methylmalonic acid and other short chain dicarboxylic acids by gas chromatography: use in prenatal diagnosis of methylmalonic acidemia and in studies of isovaleric acidemia.

We have developed a sensitive gas-chromatographic method for the determination of methylmalonic acid and other short chain dircarboxylic acids in biological samples. The method is based on the isolation of the short chain dicarboxylic acid fraction by Dowex 3 X 4 column chromatography followed by gas-chromatography analysis of these acids as methyl esters. 2-n-Pentyl-malonic acid is used as an internal standard. With this method, methylmalonic, succinic and methylsuccinic acids were consistently detected and accurately measured in urine and serum from normal subjects; the identity of these acids being verified by mass spectroscopy. The method's sensitivity permitted its used in the prenatal diagnosis of methylmalonic acidemia by measuring methylmalonic acid in urine and amniotic fluid from three pregnant heterozygous women at risk. One affected (vitamin B-12 responsive type) and two unaffected fetuses were correctly diagnosed prenatally as judged by postnatal investigations. The amount of methylmalonic acid in urine and amniotic fluid was distinctly increased (2 to 14 times normal) in the former and consistently normal in the latter two cases during the third trimester of pregnancy. Effect of prenatal therapy with large doses of vitamin B-12 was closely followed in the first case using analyses of multiple maternal urine specimens. Urinary methylsuccinic acid excretion was studied in two cases with isovaleric acidemia. It was normal in a sample from a patient in remission but was increased seven fold over control during an episode of ketoacidosis.     

Gel electrophoresis of amniotic fluid acetylcholinesterase as an aid to the prenatal diagnosis of fetal defects

The presence of absence of a specific acetylcholinesterase (AChE) band was determined by polyacrylamide gel electrophoresis on 272 second trimester amniotic fluid samples. The AChE band was absent from 176 normal samples, including seven which had been scored as false positives by alphafetoprotein (AFP) assay. It was present in all 30 samples from open neural tube defects, of which four had been scored as false negatives by AFP assay. In remaining 66 pregnancies with abnormal outcome, an AChE band was in general present when AFP was raised and absent when it was normal. However, all six cases of congenital nephrosis had raised AFP and no AChE band, while two of 30 pregnancies ending in spontaneous abortion had an AChE band and normal AFP. These results suggest that AChE electrophoresis is a valuable confirmatory technique for the early prenatal diagnosis of fetal abnormalities.     

Comparison of amino acid concentrations in amniotic fluid from fetal neural tube defective and normal pregnancies

Amniotic fluid collected during the second trimester of pregnancy was analysed for amino acid and protein concentrations. The composition of amniotic fluid from pregnancies complicated by fetal anencephaly or spina bifida was investigated for variance from normal amniotic fluid. In spina bifida the hydroxy amino acids were raised whilst the branched chain amino acids were lower in concentration. In anencephaly the total amino acid and the protein concentrations were raised, and a wider range of concentrations for most of the amino acids was apparent.     

A serine protease activity of human serum albumin towards 4-methylumbelliferyl-guanidinobenzoate (MUGB) and diisopropyl fluorophosphate (DEP): implications for the use of MUGB reactivity in amniotic fluid in prenatal diagnosis of cystic fibrosis

4-methylumbelliferylguanidinobenzoate (MUGB) reactivity in plasma from patients with cystic fibrosis and in amniotic fluid from pregnancies leading to children with cystic fibrosis, has been reported to be significantly decreased. We have so far been unable to confirm these findings and have therefore reexamined this reactivity using diisopropylfluorophosphate (DEF), another active site titrant of serine proteases. We have shown that MUGB and DFP are reacting with the same molecules in plasma and amniotic fluid. Using crossed immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis of 3H-DFP labelled plasma and amniotic fluid we have obtained strong evidence that the main contribution of MUGB and DFP reactive protein in plasma and amniotic fluid is identical to serum albumin. The use of MUGB reactivity in amniotic fluid in pregnancies at risk for cystic fibrosis must therefore be reconsidered.     

Lipid content of amniotic fluid cells

The lipid content of fetal cells was determined in 45 samples of human amniotic fluid. Free and total cholesterol were estimated using a gas chromatographic method, and glycerides were evaluated through the enzymatic assay of their glycerol content. The number of orange cells was estimated after staining with Nile Blue sulphate.

The chemically measured lipid content appeared closely related to the number of orange cells. Total cholesterol and glycerides showed a sharp increase after the 37th week of pregnancy. These tests seem to assess fetal maturity successfully, providing a further useful aid in the management of high-risk pregnancies.

The chemical determination of cell-associated lipids showed good accuracy and reliability and, when compared with the histochemical method, allowed a better evaluation of progressive lipid accumulation within the amniotic fluid cells.     

Two alpha-glucosidases in cultured amniotic fluid cells and their differentiation in the prenatal diagnosis of Pompe's disease.

A sensitive fluorometric assay utilizing 4-methylumbelliferyl-alpha-D-glucopyranoside has been developed for the determination of alpha-glucosidase. The enhanced sensitivity was achieved by increasing the solubility of the substrate with a water miscible organic solvent. With this system, cultured amniotic fluid cells were found to have two major forms of alpha-glucosidase with somewhat overlapping acidic pH optima; one with pH optimum at 4.5 is deficient in Pompe's disease (type II glycogenosis), while one with pH optimum at 6.0 is not affected in this disease. Specificity for the pH 4 form of alpha-glucosidase was achieved by exploiting the greater thermal lability of the pH 6 enzyme. The pH 6 form of the enzyme was also detectable in freshly prepared extracts of cultured fibroblasts. The procedure is direct and simple and has been applied to the prenatal diagnosis in two pregnancies at risk for Pompe's disease.     

Human serum Zn-alpha2-glycoprotein in amniotic fluid

Human serum Zn-alpha2-glycoprotein (Zn-alpha2-GP) was found to be present in the amniotic fluid in the mean concentration of 0.98 +/- 0.40 mg/100 ml, which represents about one-tenth of its concentration in the maternal serum (9.65 +/- 1.18 mg/100 ml). Its concentration in the amniotic fluid was proportional to the amniotic fluid total protein and very approximately to the maternal serum Zn-alpha2-GP. The relationship between the maternal serum Zn-alpha2GP and the maternal serum total protein as well as between the amniotic fluid total protein and the maternal serum total protein was found to be not significant. The amniotic fluid Zn-alpha2-GP as well as the amniotic fluid total protein showed some increase during gestation to reach the highest values at the end of the second trimester. At present both the origin and significance of the amniotic fluid Zn-alpha2-GP are not known.     

A rapid spot test for urinary methylmalonic acid collected on ion-exchange filter paper

We developed a rapid, accurate method to detect pathologic amounts of methyl-malonic acid (MMA) in urine samples, which can be applied to simplify and improve the accuracy of screening for methylmalonic acidemia. Urine is collected on DEAE-cellulose paper and air-dried. Six mm discs are punched from the dried paper and placed in a multi-well tray; non-anionic substances interfering with the assay are rinsed from the discs; and a color reaction using diazotized p-nitroaniline is carried out in situ on the disc.Of 407 urine samples blindly tested, all 83 samples from nine patients with methylmalonic acidemia gave positive results. None of the other urine specimens from normal individuals or patients with a variety of organic acidemias gave positive results. This method should allow accurate and inexpensive mass screening for methylmalonic acidemia in newborn or infant screening programs.     

A rapid method for assay of branched-chain keto acid decarboxylation in cultured cells and its application to prenatal diagnosis of maple syrup urine disease

A sensitive assay for measurement of branched-chain keto acid decarboxylation in small numbers of fibroblasts or amniotic cells grown in the wells of a microtitre plate using [1-14C]leucine as substrate is described. The method was applied to the amniotic cells from a pregnancy at risk for maple syrup urine disease and a heterozygous fetus predicted.     

HPLC of phospholipids in biological fluids — application to amniotic fluid for the prediction of fetal lung maturity

Because of both the advantage of speed compared with thin layer chromatography (TLC) and the dearth of high pressure liquid chromatography (HPLC) methods for phospholipid separation, it was decided to investigate the use of HPLC with a differential refractometer as detector for the separation and quantitation of amniotic fluid phospholipids required for the prediction of fetal lung maturity.A method was devised which gave results which compared well with those from TLC both in terms of quantitation and predictive value.Despite this, the method was found to lack sufficient reliability for application to the routine clinical assessment of fetal lung maturity. The method does, however, offer a good alternative to two dimensional TLC with phosphate analysis in research work involving quantitation of phosphatidyl inositol, phosphatidyl glycerol and particularly lecithin.     

Determination of phosphatidylglycerol in amniotic fluid by a simple one-dimensional thin-layer chromatography method

Phosphatidylglycerol (PG) in amniotic fluid is the second important component of lung surfactant phospholipids and may be clinically useful in assessing fetal lung maturity in utero. Although methods for PG determination are available, there are shortcomings in clinical application. We developed an alternative reliable onedimensional thin-layer chromatography (TLC) method for separating and quantitating PG in amniotic fluid. A mini-TLC plate (8 × 10 cm) was prepared from silica gel H containing 5% ammonium sulfate. The plate was first developed in tetrahydrofuron/dimethoxymethane/methanol/2N ammonium hydroxide (30.0:20.6:5.6:3.0, v/v) and then in chloroform/methanol (60:9, v/v) in the same direction. PG was clearly separated from other phospholipids and neutral lipids, even when large amounts of other phospholipids were present on the TLC plate. The density of the charred PG was directly proportional to the amount of PG up to 8 nmol. The content of PG in nmol in the specimen can be quantitated by comparing with a standard PG. Up to 10% of blood serum or 3% meconium showed no detectable PG, nor did these substances affect PG quantitation in amniotic fluid. This method is sensitive and accurate. It is also time-saving and economical.     

Mucolipidosis I: Studies of sialidase activity and a prenatal diagnosis

The characteristics of the sialidase (N-acetyl-α-neuraminidase) of human leukocytes, fibroblasts and amniotic fluid cell cultures were determined with a radioactive assay method utilizing neuramin-[³H]actitol as the enzyme substrate. Fibroblast cultures from patients with the inherited sialidase deficiency diseases including mucolipidosis I, sialidosis I and sialidosis II, juvenile type have less than 10% of normal sialidase activity using either this substrate, 2-(3′-methoxyphenyl)-N-acetyl-α-neuraminic acid, or 2′-(4-methylumbelliferyl)-Nacetyl-α-neuraminic acid. The total sialic acid content of fibroblasts and leukocytes from mucolipidosis I and sialidosis I patients is greatly elevated; this parameter is useful in establishing a diagnosis of sialidase deficiency. The sialic acid content of sialidosis II, juvenile type, with coexistent sialidase and β-galactosidase deficiencies, is only slightly elevated above normal levels. A patient with mucolipidosis I has 16% of normal neuramin-[³H]lactitol sialidase activity in his peripheral leukocytes. His parents were clearly distinguished from the normal range using leukocyte enzyme levels and a maternal aunt was identified as a possible carrier. The presence of this enzyme in amniotic fluid cell cultures, both fibroblastic and mixed cell type, makes possible the prenatal detection of these diseases. A pregnancy from a family at risk for having a child with mucolipidosis I was monitored by amniocentesis and subsequent sialidase measurement of the amniotic fluid cell culture.     

2-Methyl-3-oxovaleriansäure: ein charakteristischer metabolit bei propionacidämie2-Methyl-3-oxovaleric acid: a characteristic metabolite in propionic acidemia

The major conjugated bile acids of man, including glycine and taurine conjugates, can be separated by high-pressure liquid chromatography (HPLC). The Chromatographic column is 300 mm long with an internal diameter of 8.0 mm and is packed with Lichrosorb RP 18 (5 μm). The mobile phase is methanol-water 75: 25 (v/v) acidified to pH 2 with phosphoric acid. The eluent peaks are detected by a UV absorbance detector at a wavelength of 210 nm. The 10 conjugated bile acids are quantitatively analyzed on a single run of chromatography in less than 50 min. Deproteinization of biological samples is sufficient for the preparation of the analysis.     

Acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using 4-methylumbelliferyl palmitate.

The properties and levels of acid esterase in cultured skin fibroblasts and amniotic fluid cells were investigated using 4-methylumbelliferyl palmitate as substrate. Determinations of acid esterase activity could be made using as little as 1 microgram cell protein. Cardiolipin increased the activity 2--3 fold at the pH optimum 4.0. The apparent KM for both cell types studied was 196 micrometer without and 96 micrometer with cardiolipin. Acid esterase activity was inhibited by cyanide and thiomersal, but not by iodoacetate and N-ethylmaleimide. However activation by cardiolipin was prevented by iodoacetate, N-ethylmaleimide and also sodium chloride. Skin fibroblasts and primary amniotic fluid cells had similar levels with or without cardiolipin. A cyclic activity was found with subculture but no consistent pattern with passage. The acid esterase deficiency in Wolman's and cholesterol ester storage diseases was demonstrated with this substrate.     

Separation of acid and neutral alpha-glucosidase isoenzymes from fetal and adult tissues, cultivated fibroblasts and amniotic fluid cells by DEAE-cellulose and sephadex G-100 column chromatography.

Isoenzymes of alpha-glucosidases (EC 3.2.1.20) from various human organs and body fluids from fetuses and adults were separated by DEAE-cellulose column chromatography and gel filtration using Sephadex G-100. A minicolumn (0.35 X 2.5 cm) was used for the DEAE-cellulose column chromatography of extracts from tissues as well as cultivated cells of skin fibroblasts and amniotic fluid. The enzyme activity in the eluates was measured by the use of a methylumbelliferyl derivative as substrate and a very sensitive Microscope fluorimeter. In most tissue samples alpha-glucosidase was eluted mainly as a single peak when monitored at acid pH and as two peaks when the activity was measured at neutral pH in both columns. Another small peak representing alpha-glucosidase was found in fresh extracts of cultured cells on DEAE-cellulose columns. Neutral alpha-glucosidase especially in fibroblasts was extremely sensitive to storage at -20 degrees C. DEAE-cellulose column chromatography of plasma and amniotic fluid showed similar elution patterns of alpha-glucosidase. Differences were noticed in the elution pattern of urine from infants and adults. The tissue distribution and the different characteristics of the enzyme in samples of various origins and ages were discussed.     

Variations of the concentrations of certain glycoproteins in maternal serum, fetal serum and amniotic fluid during pregnancy (author's transl)]

The authors investigated systematically the variations during normal pregnancies of the concentrations of alpha-1-antitrypsin, orosomucoid, transferrin and alpha-fetoprotein simultaneously in maternal serum, fetal serum and amniotic fluid. The role of certain factors such as the gestational age birth weight, placental weight and pairty were studied with regard to variations in the concentrations of each of these proteins. This research permitted the definition during pregnancy of the normal concentrations for these four proteins and allowed us to learn more about protein exchanges between fetal blood, maternal blood and amniotic fluid. There exists a difference between the concentrations of alpha-1-antitrypsin and of orosomucoid found for primigravidae and for multigravidae. The role of these glycoproteins in preventing the mother from rejecting the fetus (insofar as the fetus may be considered as an allograft) is discussed.     

Diagnosis of Pompe's disease in cultured skin fibroblasts and primary amniotic fluid cells using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate.

The possible interference of neutral alpha-D-glucosidase in the diagnosis of Pompe's disease using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate for the assay of acid alpha-D-glucosidase was investigated. The pH profile of alpha-D-glucosidase in control skin fibroblasts and amniotic fluid cells showed two peaks of activity. The shape of the pH profile depended upon whether or not the extract was added to the buffer before the substrate. If extract was added to the buffer before the substrate, a greater separation was obtained between the two peaks of activity. The neutral alpha-D-glucosidase activity could be totally removed by preliminary precipitation at pH 5.0. Following acid region whilst Pompe's cells had no activity enabling a clear distinction to be made between carriers and the disease state.     

Accurate photodensitometric measurement of the lecithin: sphingomyelin ration after elimination of the acidic phospholipids from extracts of amniotic fluid specimens

Treatment of human amniotic fluid lipids dissolved in a chloroform/methanol (9:1, v/v) mixture by batchwise addition of diethylaminoethyl cellulose in a dry state proved to be an easy and rapid procedure for the removal of the acidic phospholipids which may interfere in the photodensitometric evaluation of the lecithin: spingomyelin ratio on a thin-layer chromatogram. This method was used for the measurement of the lecithin: sphingomyelin ratio in a series of normal and abnormal pregnancies. Ratios higher than expected from gestational age were observed in stressed pregnancies. A significant elevation of the ratio was also observed under treatment by dexamethasone.