Joel Hutzler

Conversion of lysine to saccharopine by human tissues

Abstract The ability of human tissues to convert lysine and α-ketoglutarate to saccharopine [ e-N-( l -glutaryl -2)- l -lysine ] has been investigated vitro. Liver is the most effective organ, though some activity could be demonstrated in several other tissues. The responsible enzyme lysine-ketoglutarate reductase (lysine:α-ketoglutarate: TPNH oxidoreductase ( e-N-[ glutaryl -2]- l -lysine forming )) has been partially purified from human liver and optimal conditions for its assay determined. There is a specific requirement for TPNH that cannot be satisfied by DPNH. A pH optimum near neutrality and inhibition by ammonium sulfate were observed. The enzyme is stable at −25°, both in tissues obtained at post-mortem and in its purified form. The assay for lysine-ketoglutarate reductase is sufficiently sensitive to be used on the limited amount of tissue obtainable by needle biopsy of the liver.

Intermittent Branched-Chain Ketonuria: Variant of Maple-Syrup-Urine Disease

MAPLE-syrup-urine disease (branched-chain ketonuria) is a genetic disorder with a well defined biochemical defect.1 Symptoms of anorexia, vomiting, hypertonicity and occasionally convulsions appear...

Enzyme activity in classical and variant forms of maple syrup urine disease

The branched-chain keto acid decarboxylase activity has been determined in skinfibroblasts grown from six subjects with classical maple syrup urine disease and six with variant forms of the disease. The level of activity in the skin fibroblasts reflects the ability of the individual to degrade the amino acids, thus providing an index of the severity of the disease. Observations on sibling pairs indicate that the level of enzyme activity and the severity of the disease are genetically determined. A 3 grade classification is proposed based on tolerance for dietary protein. The metabolic defect in all instances involved the three branched-chain amino acids, providing further support for the concept that this degradative step is under the control of a single gene.

Lysine-ketoglutarate reductase in human tissues.

Lysine-ketoglutarate reductase (saccharopine dehydrogenase (NADP+, lysine-forming) EC 1.5.1.8) from human liver has been partially purified and characterized. A spectrophotometric assay is described. The Michaelis constants have been determined for lysine (1.5-10-3 M), alpha-ketoglutarate (1-10-3 M) and NADPH (8-10-5 M). The pH optimum is 7.8. The enzyme is product inhibited. The specificity of the enzyme, response to inhibitors, pH and thermal stability are reported. Lysine-ketoglutarate reductase is present in high concentration in liver and heart, to a lesser degree in kidney and skin and in trace amounts in several other tissues. Saccharopine dehydrogenase (saccharopine dehydrogenase (NAD+, L-glutamate-forming) EC 1.5.1.9) was demonstrable only in liver and kidney. Lysine-ketoglutarate reductase reacts effectively with delta-hydroxylysine.

Familial hyperlysinemia with lysine-ketoglutarate reductase insufficiency

Fibroblasts grown in tissue culture from the skin of normal subjects have lysine-ketoglutarate reductase activity (lysine: alpha-ketoglutarate: triphosphopyridine nucleotide (TPNH) oxidoreductase (epsilon-N-[L-glutaryl-2]-L-lysine forming)). The activity of the enzyme is considerably reduced in the skin fibroblasts grown from three siblings with hyperlysinemia. The high concentrations of lysine in the blood of these patients, the previous demonstration in the intact subject of a reduction in the ability to degrade lysine, and the present demonstration of diminished lysine-ketoglutarate reductase activity, accurately define the metabolic defect and establish the saccharopine (epsilon-N-[L-glutaryl-2]-L-lysine) pathway as the major degradative pathway for lysine in the human.

Thin-layer chromatograhy and spectrophotometry of α-ketoacid hydrazones

Abstract Thin-layer chromatography has been applied to the dinitrophenylhydrazones of α-ketoacids. Rapid, effective separations are obtained, and have been used in obtaining spectrophotometric data on the dinitrophenylhydrazones of pyruvic acid and the branched-chain ketoacids including the two isomers of pyruvic acid and ketoleucine. A method is derived for calculating the relative amounts of each of the isomer dinitrophenylhydrazones of ketoleucine in a mixture of the two. a methd is described for determiniing the individual branched-chain ketoacids which could be applied to following the blood levels in maple-syrup urine disease.

Enzyme defect in skin fibroblasts in intermittent branched-chain detonuria and in maple syrup urine disease☆

Abstract The enzyme defect in maple syrup urine disease and in its varient form, intermittent branched-chain ketonuria, is demonstrable in skin fibroblast cultures derived from patients with this disease. In both instances there is considerable reduction in the ability to decarboxylate all three branched-chain keto acids. In view of the recent evidence that there are two or three branched-chain decarboxylases, each with a specific substrate, it is suggested that one gene controls the synthesis of a polypeptide that is common to the involved enzymes or to some structural or regulatory part of the enzyme complex.

The determination of pipecolic acid: method and results of hospital survey

A method is described for the quantitative assay of pipecolic acid in biological fluids using column chromatography, with acid ninhydrin for color development. The technique permits measurement of plasma pipecolic acid in normal individuals with blood samples of 1 ml. The mean value in 29 pediatric patients, aged 4 months to 17 years, hospitalized with a variety of diagnoses was 2.1 mumol/1 +/- 1.6. Newborn infants had slightly elevated plasma levels with a mean of 12 mumol/1 +/- 5.6. Among the diagnostic categories, patients with liver disease were distinctive in commonly having levels at the upper limits of normal. In some cases, the levels were exceedingly high, reaching 78 mumol/1 in one fatal case.

Multiple enzyme defects in familial hyperlysinemia.

Extract: Lysine-ketoglutarate reductase (EC. 1.5.1.8) deficiency in skin fibroblasts has been previously reported in patients with familial hyperlysinemia, providing an adequate explanation for the biochemical derangements noted clinically. In the present study, analysis of liver obtained at autopsy from a patient with familial hyperlysinemia confirmed the lysine-ketoglutarate reductase deficiency but, unexpectedly, also revealed an absence of saccharopine dehydrogenase (EC. 1.5.1.9) and saccharopine oxidoreductase activity. Skin fibroblasts from two siblings with the disease and a third patient from an unrelated family were also deficient in all three enzymes (lysine-ketoglutarate reductase, average 9%; saccharopine dehydrogenase, average 4%; saccharopine oxidoreductase, <10% of normal). The possibility that saccharopine dehydrogenase is a substrate-inducible enzyme was investigated by maintaining normal skin fibroblasts in a medium with minimal lysine concentration, and exposing hyperlysinemic fibroblasts to elevated saccharopine concentrations. There was no significant modification in saccharopine dehydrogenase activity.Speculation: Multiple enzyme defects have now been recognized in three genetic diseases, maple syrup urine disease, orotic aciduria, and hyperlysinemia, presumably arising from single mutations. It is not unlikely that additional examples will emerge as investigators increase their efforts in this direction. The simultaneous loss of enzymes may provide insights into mechanisms of control and/or the evolutionary development of enzymes.

Detection of the heterozygote in maple syrup urine disease

Parents of children with maple syrup urine disease were studied for the presence of apartial enzymatic defect. Load tests with α-ketoisocaproic acid failed to reveal any difference from normal subjects. However, there was a statistically significant reduction in decarboxylase activity in the peripheral leukocyte in the fathers as measured with radioactively labeled leucine.