H. W. Rüdiger

Rapid diagnosis of maple syrup urine disease (branched chain ketoaciduria) by micro-enzyme assay in leukocytes and fibroblasts

Abstract Two micro-enzyme assays are described for measuring directly the level of the branched chain α-keto acid decarboxylase in leukocytes and cultured fibroblasts, utilizing the conversion of [α-I-14C]keto acids as substrates. The cell material per assay is greatly reduced by using microtiter plates: 50 μl of whole venous blood or about 10000 fibroblasts, tested in monolayer, are needed per assay, and yet, accurate and reproducible results are obtained. The blood test improves and facilitates the early specific diagnosis of maple syrup urine disease in newborn infants, the test in fibroblasts assures early prenatal diagnosis.

Frequency of sister chromatid exchanges in Bloom syndrome fibroblasts reduced by cocultivation with normal cells.

SummaryCocultivation of fibroblast cells from a male patient with Bloom syndrome (BS) and a female control reduced the rate of sister chromatid exchanges in the BS cells from a mean of 54 SCE per metaphase (range 42–65) to 41 (range 24–59). Medium used to culture control cells for 48 h also reduced the rate of SCE (from 40–65 to 33–54), whereas medium used for only 24h altered the SCE rate only slightly (to 39–61). Dialyzed medium concentrate with molecular cutoff at 15,000 did not alter the SCE rate. These initial studies suggest that normal cells produce an agent, presumably lacking in BS cells, that is capable of mitigating the chromosomal manifestation of the BS mutation (bl) in bl/bl cells.

Benzpyrene-induced sister chromatid exchanges in lymphocytes of patients with lung cancer.

SummaryThe effect of benzpyrene on sister chromatid exchange was determined in PHA-stimulated lymphocytes of 18 patients with lung cancer and 11 controls without cancer or bronchopulmonary diseases. Patients and controls did not differ either with respect to the spontaneous rate of sister chromatid exchanges or in their response to the carcinogen. We conclude that individual susceptibility to lung cancer cannot be detected by an individual response to benzpyrene, at least in lymphocytes and at the chromosomal level.

Maple syrup urine disease: rapid prenatal diagnosis by enzyme assay.

This repor t describes the first an tepa r tum identif ication of a fetus homozygons for the defect ive gene of maple syrup urine disease (MSUD). The result was obta ined by direct enzyme assay in cul tured amniot ic fluid cells 15 days after the grossly sanguinolent amniot ic fluid was received. Four cases of prenata l diagnosis of maple syrup urine disease (MSUD) are on record, three of which were found to be normal (Dancis, 1972). An abnormal fetus was suspected in one, but no definite prenatal diagnosis could be established. The pregnancy went to t e rm and an abnormal child was born (Dancis, 1972).

Defective decarboxylase in branched chain ketoacid oxidase multienzyme complex in classic type of maple syrup urine disease

SummaryEnzyme kinetic studies on tissue extract from a newborn child who had died from the classic type of maple syrup urine disease (MSUD) revealed an altered decarboxylase moiety of the branched chain ketoacid oxidase multienzyme complex. Of two kinetically different decarboxylase components present in normal human controls the one with higher affinity for the substrate α-ketoisovalerate was absent in our patient.ZusammenfassungEs wurden enzymkinetische Untersuchungen des verzweigtkettigen α-Ketosäureoxidase-Komplexes in angereicherten Gewebeextrakten eines neugeborenen Kindes beschrieben, das an sogenannter klassischer Ahornsirupkrankheit gestorben ist. Während in normalen Kontrollen zwei kinetisch unterscheidbare Komponenten mit unterschiedlicher Substrataffinität nachweisbar sind, wurde im Gewebe des Patienten nur die Komponente mit geringerer Affinität zur α-Ketoisovaleriansäure gefunden. Dieses Ergebnis deutet darauf hin, daß bei der klassischen Ahornsirupkrankheit das Teilenzym Decarboxylase im Multienzymkomplex der verzweigtkettigen Ketosäureoxidase defekt ist.

Studies on the optimal cooling rate for freezing human diploid fibroblasts

Abstract Ampoules containing each 1 ml of Dulbeccos Modified Eagle Medium with 16.6% fetal calf serum and 10% dimethylsulfoxide were insulated in various ways and placed into different cooling boxes. The resulting cooling velocities of the medium ranged from about 0.7 to 102 °C/min. As revealed by cellular attachment in recovery cultures, human diploid fibroblasts cooled at about 1.5 to 4.5 °C/min to − 78 °C prior to storage in liquid nitrogen showed an optimal survival of about 60%. Survival was about 25% at cooling rates of 0.7 and 19 °C/min, respectively. The optimal cooling rate was achieved by insulating the freezing ampoules with 1 to 3 closed vessels and placing them into a dry ice chest, or into a dry ice/ethanol bath.

Impaired insulin-induced RNA synthesis secondary to a genetically defective insulin receptor

SummaryThe stimulation of glucose uptake and RNA synthesis by insulin was studied in cultured fibroblasts from patients with an inherited affinity defect of the insulin receptor. Additional cell cultures were set up of two patients with Alstrøm syndrome, a genetic disease with insulin resistance but normal insulin receptor, and of eight healthy individuals. In receptor-defective patients we found a corresponding defect of the insulin-mediated stimulation of RNA synthesis but a normal stimulation of glucose uptake. We conclude that both postreceptor effects are controlled by different insulin-directed mechanisms.

Toxicity of antibiotics on cultured human skin fibroblasts

SummaryHuman diploid fibroblasts cultured in Dulbeccos Modified Eagles medium (DME) were exposed to different concentrations of 15 antibiotics to determine the limiting toxic concentration. The number of cells surviving after antibiotic treatment was given as the index of toxicity. No visible chromosomal damage could be detected when half the maximal toxic concentration was applied. The maximum limiting concentration was found to be the same for both the preconfluent and postconfluent phases.Zusammenfassung15 Antibiotica wurden in verschiedenen Konzentrationen menschlichen diploiden Fibroblasten, gezüchtet in Dulbeccos Modified Eagles medium (DME), zugesetzt. Die Grenze der toxischen Konzentration wurde für jedes Antibioticum durch Zellzählungen in unabhängigen Versuchen bestimmt. Die für den antimikrobiellen Einsatz verwendbaren Maximalkonzentrationen wurden angegeben als die Hälfte der minimalen toxischen Konzentrationen. Keines der getesteten Antibiotica führte bei diesen Konzentrationen zu chromosomalen Aberrationen. Die toxische Grenze war jeweils gleich für Zellen der präkonfluenten und postkonfluenten Phase.

Evaluation of a heterozygote test for maple syrup urine disease in leucocytes and cultured fibroblasts

SummaryMethods are given in detail to assay branched chain keto acid oxidases in native leucocytes and fibroblasts. In peripheral blood these enzymes are located preferentially in lymphocytes.The intraindividual variation of enzyme activities in leucocytes is reduced by correcting for the number of lymphocytes. In contrast, interindividual variation remains unchanged. Consequently, an overlap between enzyme activities of control persons and heterozygotes for classic maple syrup urine disease still exists. For explanation multiple alleles and influence of genetic background on enzyme activities are invoked.Arguments are given for the simultaneous defect of the three branched chain keto acid oxidases in classic maple syrup urine disease.Furthermore some new observations on the intermittent type of maple syrup urine disease are given.Tests for heterozygosity in fibroblasts are complicated because of environmental influences in cultures which are not fully understood at present. However, the enzymatic defect is clearly demonstrated in fibroblasts of patients with the classic type and the intermittent type of maple syrup urine disease.ZusammenfassungDie Methoden zur Testung der Oxidasen für die verzweigtkettigen α-Ketosäuren in Leukocyten und Fibroblasten werden beschrieben. Im peripheren Blut sind diese Enzyme bevorzugt in den Lymphocyten lokalisiert.In den Leukocyten wird die intraindividuelle Variation der Enzymaktivitäten durch Berücksichtigung des Differentialblutbildes verringert. Die interindividuelle Variation bleibt dagegen unverändert. — Für die Enzymaktivitäten von Normalpersonen und Eltern von Patienten mit klassischer Ahornsirupkrankheit bleibt damit ein Überlappungsbereich bestehen. Als mögliche Erklärung werden multiple Allelie und multifaktorielle Determinierung von Enzymaktivitäten diskutiert.Bisher gewonnene Ergebnisse lassen vermuten, daß bei der klassischen Form der Ahornsirupkrankheit alle drei Oxidasen für die verzweigtkettigen α-Ketosäuren defekt sind. Neuere Untersuchungen über die intermittierende Form der Ahornsirupkrankheit werden mitgeteilt.Die Erkennung von Heterozygoten in Testen mit Fibroblasten ist erschwert, da die Abhängigkeit der Aktivität der α-Ketosäure-Oxidasen von den Kulturbedingungen noch nicht genügend geklärt ist. Es ist dagegen möglich, Patienten mit der klassischen und der intermittierenden Form der Ahornsirupkrankheit durch enzymatische Teste an Fibroblasten zu erkennen.

Enhancement of amniotic fluid cell growth in culture

SummaryA factor which promotes growth of diploid fibroblasts and amniotic fluid cells was prepared from human urine. The addition of up to 10% of this growth factor together with 20% fetal calf serum to the culture medium was found to be optimal for stimulation of amniotic fluid cell growth. Microtest plates have several advantages compared with other methods to initiate an amniotic cell culture.ZusammenfassungDas Wachstum von Amnionzellen und diploiden Fibroblasten wird durch Konzentrat von menschlichem Urin stimuliert. Ein optimales Anwachsen von Amnionzellen wurde erreicht, wenn das Kulturmedium mit 20% fetalem Kälberserum und bis zu 10% Urinkonzentrat versetzt wurde. Das Anlegen einer Amnionzellkultur in Mikrotestplatten hat Vorteile gegenüber anderen Methoden.