Howard Tieckelmann

Intermittent non-ketotic dicarboxylic aciduria in two siblings with hypoglycaemia: An apparent defect in β-oxidation of fatty acids

Two siblings with intermittent hypoglycaemia, lethargy and coma associated with fatty infiltration of the liver are reported. Urine contained C6 to C14-dicarboxylic acids.

Separation of reduced disaccharides derived from glycosaminoglycans by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the analysis of reduced unsaturated disaccharides derived from enzymatic digestion followed by reduction with sodium borohydride of chondroitin sulfates, dermatan sulfate, heparan sulfate and heparin is described. This method is well suited for the HPLC analysis of glycosaminoglycans (GAGs) because the possibility of obtaining anomeric forms of unsaturated disaccharides is eliminated with provides a major advantage for quantitation. This procedure is more sensitive than existing HPLC methods for the determination of enzymatic degradation products from GAGs. In particular, the resolution of disaccharide products from heparan sulfate is improved after reduction. The applicability of this method for the determination of GAGs in biological samples is demonstrated.

Rapid and sensitive determination of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography

The separation and quantitative analysis of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography (HPLC) are described. The substituted unsaturated disaccharides which result from digestion of chondroitin sulfates with chondroitinase are quickly separated on polar absorbents such as silica gel. The UV absorption properties of these unsaturated disaccharides permit UV measurement with detection limits of approximately 100 ng. Their separation by HPLC facilitates the use of enzymatic methods for the determination of chondroitin sulfates A, B and C. The potential of this method in clinical application is demonstrated by quantitative assays of glycosaminoglycans from a normal urine and urine from a patient with Hunter syndrome. The results are consistent with amount of isomeric chondroitin sulfates found in comparable urines by others.

Detection of δ-aminolevulinic acid, porphobilinogen and porphyrins related to heme biosynthesis by high-performance liquid chromatography

Abstract Two new high-performance liquid chromatographic methods are described for the quantitative determination of porphyrins and their precursors. In our method, sub-nanomole quantities of prophyrins, δ-aminolevulinic acid and porphobilinogen derivatized with o -phthalaldehyde were injected onto a C 18 reversed-phase column and eluted with 0.1 M monobasic sodium phosphate—methanol—tetrahydrofuran (4:6:3) and detected with a spectrofluorometer. A second reversed-phase system using methanol—tetrahydrofuran— 22 m M acetate buffer (15:6:11) was also developed.

Quantitative determination of porphyrins, their precursors and zinc protoporphyrin in whole blood and dried blood by high-performance liquid chromatography with fluorimetric detection

Methods for the determination of porphyrins, delta-aminolevulinic acid (ALA), porphobilinogen (PBG) and zinc protoporphyrin of heme biosynthesis in whole blood and dried blood are described. Erythrocyte porphyrins and the precursors ALA and PBG were extracted from whole blood (50 microliter) with 0.3 ml of methanol and 1.5 M hydrochloric acid (2:1, v/v). Zinc protoporphyrin was extracted with an acetone-pyridine-Sterox solution. Other major interfering metabolites were removed by centrifugation. An aliquot of the supernatant was injected onto the reversed-phase C18 column for detection of porphyrins with excitation wavelength at 405 nm and emission wavelength at 630 nm. The mobile phase was 0.1 M phosphate-methanol-tetrahydrofuran (18:30:16, v/v/v), pH 5.38. The ALA and PBG were derivatized with o-phthalaldehyde before injection. The detection excitation wavelength occurred at 330 nm and the emission wavelength at 418 nm. The mobile phase was 0.1 M phosphate-methanol (7.5:5), pH 3.38. For the dried blood specimen of filter paper, two 0.64-cm discs punched out from the blood-impregnated filter paper were placed in a test tube containing 200 microliter of 0.9% saline for 60 min or longer at room temperature and then treated as whole blood.

Enzymatic studies of urinary isomeric chondroitin sulfates from patients with mucopolysaccharidoses. The application of high performance liquid chromatography.

The high-performance liquid chromatographic (HPLC) method for the determination of unsaturated sulfated disaccharides is a comprehensive and reliable method which expedites ensymatic studies of isomeric chondroitin sulfates. Responses for these unsaturated disaccharides derived from urinary chondroitin sulfates were linear from 100 ng to 10 micrograms injected and good quantitation was obtained for 25 microliters or less of samples placed on the column. This method which is more sensitive and accurate than methods now being used has considerable potential for the chemical diagnosis of patients with mucopolysaccharidoses and for the clarification of glycosaminoglycan structure. The isomeric chondroitin sulfates in urines from patients with mucopolysaccharidoses were studied by enzyme digestion with chondroitinases followed by HPLC determination of the sulfated unsaturated disaccharides produced. Evaluation by HPLC of the unsaturated 4-sulfated disaccharide produced by digestion of the urinary GAG with chondroitinases ABC and AC revealed rapidly and quantitatively the large amounts of dermatan sulfate present in Hurler, Hunter, and Maroteaux-Lamy urines. Chondroitin 4-sulfate predominated in Sanfilippo urinary isomeric chondroitin sulfates whereas chondroitin 6-sulfate and chondroitin 4-sulfate were shown to be present in nearly equal amounts in Morquio urine. An oversulfated chondroitin sulfate was detected in small amounts in some of these urines. This was demonstrated by the detection of an unsaturated disulfated disaccharide after digestion with chondroitinase ABC but not with chondroitinase AC.

Precursors of the pyrimidine and thiazole rings of thiamine

Abstract An investigation of the precursors for de novo synthesis of thiamine was carried out by pulsing log phase cultures of Bacillus subtilis with labeled substrates. It was concluded that the pyrimidine ring is derived primarily from formate, acetate and aspartate, and the thiazole ring totally from alanine and methionine. A scheme is presented suggesting the specific carbons of thiamine which originate from each precursor molecule.

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography.

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in combination with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10(3) concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.

The application of high-performance liquid chromatography in enzymatic assays of chondroitin sulfate isomers in normal human urine

A study of the urinary excretion of isomeric chondroitin sulfates in normal individuals by high-performance liquid chromatographic (HPLC) determinations of the unsaturated disaccharides produced by digestion with chondroitinases is described. The composition of the HPLC mobile phase was systematically varied in order to select the optimal conditions for separation. The data show that chondroitin 4-sulfate is the major component of the chondroitin sulfate isomers in normal urine, and that chondroitin 6-sulfate is a lesser component. It is also evident that dermatan sulfate is present in small quantities in normal urine.

Phenylalanine metabolism: the production of phenylacetaldehyde by a Proteus species.

Abstract In the Proteus suspension under study it was shown that phenylalanine, 2 phenyl pyruvate, phenyl acetate, mandelic acid, and phenyl lactate at a 12 μmoles/ml, level were noninhibitory to glucose oxidation if glucose was added at zero time. Benzoic acid and benzaldehyde at a level of 15 μmoles/ml, were somewhat inhibitory. Metabolism of phenylalanine and phenyl pyruvate by the Proteus suspension produced a volatile, sweet-smelling aromatic substance, the production of which was coincident with complete inhibition of glucose oxidation. This substance has been identified as phenylacetaldehyde. Added phenylacetaldehyde, 0.3 μmole/ml., completely prevented glucose oxidation. This compound completely prevented Proteus growth at a 4 μ-moles/ml. level. Although human metabolism and Proteus metabolism of phenylalanine are not necessarily alike, it is tempting to speculate that one of the toxic products of the Proteus suspension and of phenylketonurics is phenylacetaldehyde.