Klaus O. Gerhardt

Quantitative enzymatic hydrolysis of tRNAs : Reversed-phase high-performance liquid chromatography of tRNA nucleosides☆

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme--substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis--HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.

The determination of part-per-billion levels of citric and nitrilotriacetic acids in tap water and sewage effluents.

Abstract Citric and nitrilotriacetic acids can be determined at the 1–10,000 p.p.b. 1 levels in aqueous systems ranging from tap water to sewage effluents by use of anion-exchange clean-up, derivatization with butanol—HCl and gas chromatography. A variety of metals present at legal tolerance limits do not interface. The two esterfied acids separate well on a special gas chromatographic phase; however, citric acid can also be separated from nitrilotriacetic acid by ion exchange prior to derivatization, if so desired.

High-performance liquid chromatographic analysis of biogenic amines in biological materials as o-phthalaldehyde derivatives☆

A remarkably sensitive, simple and selective reversed-phase high-performance liquid chromatographic (HPLC) method has been developed, allowing, for the first time, the direct measurement of histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin and tyramine in a single sample of plasma (2 ml), tissue (0.2 g), or urine. The biogenic amines were modified by pre-column derivatization with o-phthalaldehyde which stabilizes the molecules, aids in extraction, and improves HPLC detection at the nanogram level. To minimize losses during the sampling procedure a careful collection procedure was designed. We developed a simple sample cleanup in which the samples were thawed, neutralized with KOH, immediately derivatized, extracted into ethyl acetate (EtOAc) and then chromatographed by HPLC. The derivatives were stable in EtOAc for more then 24 h. Interfering amino acids were removed from the EtOAc by partitioning twice with Na2HPO4 buffer (pH 10.0). Complete separation was achieved in ca. 60--90 min on a muBondapak phenyl column using a stepwise gradient of acetonitrile and/or methanol-phosphate buffer (pH 5.1). A variable wavelength fluorometer with a 5-microliter flow-cell was used (excitation 340 nm; emission 480 nm). Linearity ranged from 200 pg to 50 ng onto the column. Precision (R.S.D.) for retention times was 1% and for derivatization and injection 2.5%. Recoveries of the seven biogenic amines from plasma spiked with 25 ng/ml averaged 70%, with a relative standard deviation of 6%. Separation studies were also done using a muBondapak C18 column. The effects of various eluents are presented. Gas-liquid chromatography was also investigated but lacked the sensitivity achieved by HPLC. The HPLC method is used routinely for the determination of biogenic amines in plasma from pigs with malignant hyperthemia and thermally stressed bovine. Significant differences in levels of biogenic amines were noted between stressed and non-stressed animals. Data on rat brain tissue samples were compared with the trihydroxyindole method and canine heart tissue was analyzed for ventricular norepinephrine and dopamine. Application of the method to urine from normal persons and a patient with a brain tumor has been demonstrated.

Rapid microdetermination of fatty acids in biological materials by gas-liquid chromatography.

Total and free fatty acids in general ranging from lauric to nervonic acid were separated and quantitated based on an internal standard method as methyl esters by “on column” methylation with trimethyl-(α,α,α-trifluoro-m-tolyl) ammonium hydroxide (TMTFTH) in a gas chromatographic system. This study represents an application of a method published by MacGee and Allen and a change to an internal standard technique. For the determination of the total fatty acids the sampls were saponified with KOH-CH3OH, acidified with H2PO4, and then the fatty acids were extracted into hexane. An aliquot of the hexane extract was then extracted with TMTFTH and chromatographed. For determination of free fatty acids the sample was acidified with H3PO4, immediately extracted with hexane and processed as described earlier. The relative standard deviation of 1.4 to 4.2% illustrates the precision of the method and the recovery of the fatty acids ranged from 88.5 to 100.5%. This method was applied to the determination of fecal fatty acids in conjunction with an interdepartmental study on “High protein diet in colon cancer” at the University of Missouri. In addition, the applicability of the analytical procedure (with small modifications) was shown for a wide variety of biological materials (serum, milk, skin tissue, fungal spores, food homogenates, beef tissues, and tumor cell cultures). The analyses were performed on different gas chromatographs by different analysts.

Gas-liquid chromatography-mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers.

tert.-Butyldimethylsilyl ethers of secondary hydroxy fatty acid methyl esters (tBDMS-O-FAMEs) produce stable derivatives amenable to gas-liquid chromatography (GLC) and mass spectrometry (MS). Derivatives produce prominent molecular mass minus 57 [M-57]+ fragment ions and unique marker fragment ions indicating the location of the secondary hydroxyl groups along the aliphatic chain from the omega-2 carbon to carbon numbers 5 from the carboxylic terminus, in addition to yielding information regarding carbon chain length, and degree of unsaturation. The tBDMS-derivatives of C-2, C-3 hydroxy fatty acids and the unique GLC-MS data of gamma- and delta-lactones are also presented. Though several tBDMS-O-FAMEs with centrally located hydroxyl groups were not chromatographically resolved, the combination of GLC retention times and monitoring of key diagnostic fragment ions of each tBDMS-derivative, when applied to mixtures containing all hydroxy isomers of palmitic through arachidic acid methyl esters, and to several monounsaturated, monohydroxylated fatty acid methyl esters, allowed for their unambiguous identification. Coupled with derivative stability, permitting their purification and concentration, this method was applied to the identification of trace lipids isolated from bovine skim milk which contained a complex mixture of hydroxy fatty acids of which 19 were newly identified.

Plasma levels of norepinephrine and epinephrine during malignant hyperthermia in susceptible pigs

Malignant hyperthermia (MH) is a genetic disease of man, swine, dogs, cats, and horses. The syndrome is normally triggered by inhalational anesthetics or the administration of depolarizing muscle relaxants such as succinylcholine or various environmental stress factors. We have used the MH-susceptible pig as an animal model to study the hormonal changes developing during this highly lethal syndrome. High-performance liquid chromatography with electrochemical detection was used for the quantitation of the plasma levels of norepinephrine and epinephrine during MH. This research presents evidence that the rapid release of massive quantities of norepinephrine (up to 108 ng/ml) into the blood stream occurs simultaneously with the initiation of tachycardia which is the herald signal of the onset of MH. Norepinephrine levels exceed epinephrine by a 4:1 ratio early in the syndrome. Even pigs with MH which do not develop the muscle rigor phase have high levels of circulating norepinephrine. Tachycardia, pulmonary hypertension, increased venous oxygen desaturation, and increasing core temperature develop as the syndrome progresses.

Chemical analysis of peach extrafloral nectary exudate

Abstract Analyses of peach leaf nectary exudate confirmed the presence of seven carbohydrates. These included two amino sugars and inositol which have not been previously reported in nectary exudate of plants. Seventeen amino acids, and seven fatty acids were also detected. The major carbohydrate fraction (in decreasing order of concentration) consisted of fructose, glucose and sucrose.

Pre-column derivatization and high-performance liquid chromatography of biogenic amines in blood of normal and malignant hyperthermic pigs☆

A sensitive, selective, pre-column derivatization method was used with high-performance liquid chromatography to measure norepinephrine, dopamine and serotonin in plasma from normal and malignant hyperthermic (MH) pigs. Samples were carefully collected from control and stressed animals under halothane anesthesia. Using a simple extraction method involving pre-column derivatization with o-phthaladehyde and ethyl acetate partitioning, the samples were chromatographed in less than 50 min. Norepinephrine was found to be elevated in MH pigs as the syndrome progressed, reaching levels eight-fold greater than control pigs under anesthesia. These experiments provide some evidence for our hypothesis that a failure to metabolize excess norepinephrine may be one of the key metabolic defects in causing the pathophysiology of the malignant hyperthermia-stress syndrome. The application of our chromatographic method in animal and human tests may provide a pattern of biogenic amine types and levels that could be diagnostic in identifying susceptible humans and carrier animals.

The negative alkali flame detector response

Abstract Under certain conditions, the alkali flame detector will give negative response (inverted peaks) for organic compounds containing halogen, nitrogen, or other elements. With a modification of the alkali flame detector particularly suited for such a study, the range and magnitude of negative response was defined in terms of alkali bead bore, hydrogen flow, carrier gas flow, electrode height, and other parameters. Organic compounds containing chlorine, bromine, iodine, and nitrogen, were used as test substances; an organophosphate was studied under the same conditions for purposes of comparison. Cl, Br, I, N, and C can show either positive or negative response and each can be distinguished from the others by proper choice of parameters. Both halides and carbon compounds show stronger response in the negative mode than in the positive mode. The use of two different carrier gases, nitrogen and helium, did not cause significant differences, except in its effect on the response of nitrogen compounds. In general, a large bead bore, a high hydrogen supply and/or a low carrier gas flow favor negative response. These are also the conditions which establish a large area of contact between flame and alkali surface and, consequently, a large background current. In order to obtain reproducible response of adequate magnitude, the Rb2SO4 salt surface must be smooth and homogenous. The described detector functions can be used for qualitative microanalysis of hetero-organic compounds by gas—liquid chromatography.

Storage protein in hereditary ceroid-lipofuscinosis contains S-methylated methionine

Hereditary ceroid-lipofuscinoses are neurodegenerative disorders characterized by the accumulation in numerous tissues of a storage material with lipofuscin-like fluorescence properties. Investigations were undertaken to determine the chemical nature of this storage material isolated from the cerebral cortex of human subjects with the late infantile form of the disease. The storage material was mainly protein that consisted of a mixture of polypeptides ranging in apparent molecular weight from 13 to 67 kDa. Protein-bound fluorophores apparently were largely responsible for the autofluorescence of the storage bodies. The disease-related storage body protein was rich in S-methylmethionine [(3-amino-3-carboxypropyl) dimethyl sulfonium ion], an amino acid that does not normally occur in animal proteins. Methylation of proteins to form this unusual charged amino acid may impair proteolytic degradation or other aspects of protein metabolism, and account for the accumulation of protein-filled inclusions in cells of individuals with ceroid-lipofuscinoses. Similar amino acid modifications that block proteolysis could be involved in age pigment accumulation.