Abraham Saifer

Comparative study of various extraction methods for the quantitative determination of free amino acids from brain tissue

Abstract Seven published extraction methods for the quantitative determination of the amino acids of biological fluids and tissues were applied to brain tissue and the experimental data analyzed statistically. All procedures employing organic solvents, i.e., 75% ethanol, acetone-HCl, and chloroform-methanol, yielded low values of the basic amino acids as compared to perchloric acid extraction. Of the acid extraction methods, trichloroacetic, sulfosalicylic, and perchloric acids gave essentially similar values for all but a few of the amino acids. Picric acid extracts demonstrated similarities to the organic solvents in giving low basic amino acid values as well as showing significant differences in a number of other amino acid values as compared to perchloric acid extracts. On the basis of these findings, perchloric acid extraction was judged to be the most suitable extraction method for free amino acid analysis of body tissues or fluids. When used in conjunction with a single-column ion-exchange resin automated technique and lithium citrate buffer elution, it makes possible the separation and quantitative analysis of all the amino acids, including glutamine and asparagine, usually found in biological fluids and tissues.

Identification of Tay-Sachs disease carriers by acrylamide gel electrophoresis.

Abstract 1. 1. For carrier detection in the general population, a simple, rapid quantitative acrylamide gel electrophoretic technique has been developed which employs a fluorogenic (umbelliferyl) substrate and automatic fluorimetric scanning of the gels. 2. 2. With the procedure the leukocytes of 6 Tay-Sachs disease carriers had a range of N-acetylhexosaminidase A values of 33–52% (mean = 49% A) while the normal range was 62–76% (mean = 66% A) with no overlap between the two groups. 3. 3. The presence of two isoenzymes, A and B, of N- acetyl -β- d - hexosaminidase (β-2-acetamido-2-deoxy- d -acetamidodeoxyglucohydrolase, EC 3.2.1.30) in the leukocytes of normal individuals (66% A/34% B) and absence of the A form in leukocytes of 12 Tay-Sachs disease patients has been confirmed.

Screening for Tay-Sachs disease in utero using amniotic fluid.

Summary The amniotic fluid from 38 control and 5 heterozygote TSD mothers were analyzed by acrylamide gel electrophoresis and heat fractionation for the percent of hexosaminidase A activity. This study indicates that amniotic fluid, when analyzed by acrylamide gel electrophoresis, can be used for the prenatal diagnosis of TSD. ADDENDUM Since this paper was submitted for publication, the amniotic fluids of 35 additional normal (non-Tay-Sachs) pregnant women, 3 pregnant Tay-Sachs mothers (predicted normal) and 2 pregnant Tay-Sachs mothers (predicted Tay-Sachs) were analyzed for total hexosaminidase and hexosaminidase A content with both the heat fractionation and acrylamide gel procedures. The results obtained extend and confirm the findings reported in this paper.

Amino acid composition of monomeric and polymeric human serum albumin

Abstract All previous amino acid analyses of crystalline human serum albumin (HSA) have been performed on preparations which have been shown by gel electrophoresis to contain a number of polymeric forms. Monomeric albumin was separated from its higher M.W. components by means of a Sephadex G-150 column with Tris NaCl buffer at pH 8.1 using crystalline human albumin (Kabi) as the starting material. The physicochemical characteristics of the monomeric and polymeric fractions were investigated by means of acrylamide gel electrophoresis and immunoelectrophoresis. Their amino acid composition was determined by automated ion-exchange chromatography. Although no significant differences were found between the original albumin sample and its monomer, some of the analytical data obtained differed from those of other investigators. The polymeric fraction showed some significant differences in amino acid composition from the monomer that require further verification.

Rapid test for the detection of tay-sachs disease heterozygotes and homozygotes by serum hexosaminidase assay

Abstract Total serum hexosaminidase activity is generally measured quantitatively by the amount of fluorescent product formed by action of its various isoenzymes on the 4-methyl-umbelliferyl-β-D-N-acetylglucosaminide substrate at pH 4.5. Only the heat labile form of the enzyme (Hex A) is inactive in fluids and tissues of Tay-Sachs children. Known carriers (parents) have serum Hex A values about sol1 2 of those found for non-carriers. Carrier detection tests, presently utilized for the mass screening of the Ashkenazi Jewish population, are based either on prolonged heat inactivation or electrophoretic separation of Hex A from its other serum isoenzymes. We found that serum Hex A can also be inactivated by 5 min incubation at pH 2.8 ± 0.05 and 37°. This finding led to a simple, rapid, quantitative assay for Hex A which was applied to 29 sera (14 normal, 8 carrier, 4 pregnancy and 3 Tay-Sachs disease). Except for the pregnancy and carrier groups, there was no overlap in serum Hex A values for any of the other categories. The results by acid pH inactivation correlated well with those obtained by the heat denaturation procedure. The greater potential of the acid inactivation method for automated systems, suitable for mass screening of carriers, is presently under development.

Continuous UV monitoring of fluorogenic substrates: I. Kinetic analysis of N-acetyl-β-d-hexosaminidases☆

Abstract Determination of the uv absorption spectra of 4-MUF-GlcNAc and 4-MUF showed them to differ significantly at 350 nm. This finding was applied to the enzymatic hydrolysis of fluorogenic 4-MUF-O-substituted substrates with a continuous, spectrophotometric assay procedure. With the use of this automated technique, selected kinetic properties, i.e., Ki, Km, and V, of “purified” liver N-acetyl-β- d -hexosaminidases A and B and of crystalline Jack bean meal hexosaminidase were determined and found to be in close agreement with previousy published data obtained by conventional single point assay methods. The described technique is fast, accurate, and permits instantaneous measurements of the kinetic properties of certain enzymes implicated in a number of genetic disorders.

The free amino acids in maternal and fetal extracellular fluids collected during early pregnancy

Abstract Quantitative free amino acid levels of amniotic fluid, maternal and fetal sera, and fetal urine were determined in 9 early pregnancies. Statistical analysis indicated a positive correlation between amino acid levels in amniotic fluid with those in fetal serum and urine. These data suggest an exchange mechanism between amniotic fluid and fetal serum, in addition to placental exchange, with fetal urine acting as a diluent of amniotic fluid. Analogue studies in 2 pregnancies indicate a high turnover of amino acids in maternal serum with a slower rate in amniotic fluid (t½ = 8 ± 1 days). It is postulated that genetic disorders which result in high concentrations of metabolites in fetal serum could be detected by amniotic fluid analysis.

Pre- and Postnatal Detection of Tay-Sachs Disease. A Comparative Study of Biochemical Screening Methods

Tay-Sachs disease, an invariably fatal cerebral storage (GM2-ganglioside) disorder inherited as an autosomal recessive trait, is presently the only such genetic disease which possesses all three criteria necessary to prevent the birth of homozygotes. These criteria are:- (a) The characterization of a well-defined, high risk group in the general population which carries the defective gene, e.g., Ashkenazi Jews in the United States (1). (b) A simple, quantitative biochemical test, preferably based upon the analysis of the deficient enzyme, which will permit the detection of the heterozygotes in the normal population and the isolation of the high-risk carrier-couples, e.g., the assay of hexosaminidase A in serum (2) or white blood cells (3,4) with fluorimetric procedures. (c) The prenatal diagnosis of the disease by enzymatic analysis of the amniotic fluid or cells obtained from the fetuses of carrier-couples sufficiently early to give the parents the choice of safely terminating the pregnancy(5).

Rapid Screening Methods for the Detection of Inherited and Acquired Aminoacidopathies

Publisher Summary Two new developments in clinical medicine have made the early recognition of inborn metabolic errors in the clinical chemistry laboratory of increased importance. Utilization of dietary regimens deficient in the metabolites normally acted upon by the missing enzyme serves to ameliorate the more serious consequences of the disease, for example, low-phenylalanine diets in cases of phenylketonuria. The availability of amniotic fluid early in pregnancy by amniocentesis has made possible the prenatal diagnosis of genetic disease in those conditions in which the accumulated substance or missing enzyme is known. This approach is discussed in this chapter that helps to determine whether pregnant women, who have previously given birth to children with Tay–Sachs disease, are carrying an abnormal fetus by analyzing the amniotic fluid for the missing enzyme—that is, β-D-N-acetylhexosaminidase A. An early diagnosis in this situation is essential if the mother is to have a choice of ending the pregnancy or carrying the fetus to term.

Infrared spectra of the biologically important DNP and PTH amino acids

Abstract The infrared spectra of the DNP and PTH derivatives of 27 amino acids that are important in the study of biological materials were determined. These spectra include nine DNP and five PTH amino acid derivatives not previously reported in the literature. In addition to the usual qualitative assignment of the wavelength of spectral absorption bands in the 2 to 15 μ region, a quantitative study of the intensities of the major bands was made. This quantitative approach furnishes an additional parameter by means of which structurally related amino acids can be readily differentiated. The infrared spectra of DNP and PTH amino acids were shown to be a useful tool for the determination of the N -terminal residues of various peptides and proteins especially when used in conjuction with thin-layer chromatography.