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Biomedical subjects

C R Roe

Publications and source records attributed to C R Roe.

At least 55 records · Page 3Linked to original sources
PubMed1993

Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry.

A new method for quantifying specific amino acids in small volumes of plasma and whole blood has been developed. Based on isotope-dilution tandem mass spectrometry, the method takes only a few minutes to perform and requires minimal sample preparation. The accurate assay of both phenylalanine and tyrosine in dried blood spots used for neonatal screening for phenylketonuria in North Carolina successfully differentiated infants who had been classified as normal, affected, and falsely positive by current fluorometric methods. Because the mass-spectrometric method also recognizes other aminoacidemias simultaneously and is capable of automation, it represents a useful development toward a broad-spectrum neonatal screening method.

Humans↗
PubMed1993

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a disorder of fatty acid catabolism, with autosomal recessive inheritance. The disease is characterized by episodic illness associated with potentially fatal hypoglycemia and has a relatively high frequency. A rapid and reliable method for the diagnosis of MCAD deficiency is highly desirable. Analysis of specific acylcarnitines was performed by isotope-dilution tandem mass spectrometry on plasma or whole blood samples from 62 patients with MCAD deficiency. Acylcarnitines were also analyzed in 42 unaffected relatives of patients with MCAD deficiency and in other groups of patients having elevated plasma C8 acylcarnitine, consisting of 32 receiving valproic acid, 9 receiving medium-chain triglyceride supplement, 4 having multiple acyl-coenzyme A dehydrogenase deficiency, and 8 others with various etiologies. Criteria for the unequivocal diagnosis of MCAD deficiency by acylcarnitine analysis are an elevated C8-acylcarnitine concentration (> 0.3 microM), a ratio of C8/C10 acylcarnitines of > 5, and lack of elevated species of chain length > C10. These criteria were not influenced by clinical state, carnitine treatment, or underlying genetic mutation, and no false-positive or false-negative results were obtained. The same criteria were also successfully applied to profiles from neonatal blood spots retrieved from the original Guthrie cards of eight patients. Diagnosis of MCAD deficiency can therefore be made reliably through the analysis of acylcarnitines in blood, including presymptomatic neonatal recognition. Tandem mass spectrometry is a convenient method for fast and accurate determination of all relevant acylcarnitine species.

Acyl-CoA Dehydrogenase↗
PubMed1992

The effect of an amino acid-lowering diet on the rate of melphalan entry into brain and xenotransplanted glioma.

Melphalan (L-phenylalanine mustard, L-PAM, alkeran; molecular weight, 305,000) is transported across tumor cell membranes and the blood-brain barrier by the large neutral amino acid (LNAA) transport system. Normally, plasma LNAA levels are high enough and the affinity low enough that this system does not transport much melphalan into the brain. However, plasma amino acids can be reduced by fasting and protein-free diet. We used this method to reduce competition and to increase melphalan transport into brain tumors. In nude mice fasted for 12 h and then fed a protein-free diet for 2 and 6 h, mean plasma LNAA levels were 46% and 42% of control values. Nude mice with xenotransplanted D-54MG human gliomas were used to study tissue distribution and uptake kinetics of [3H]melphalan in a control group and a diet group (after a 12-h fast and 2 h of a 0% protein diet). The K1 (blood-to-tissue transfer constant) of melphalan, determined by graphical analysis and by nonlinear fitting to a 2-compartment model, was higher in the diet group in all tumor regions except the necrotic center of subcutaneous tumors; the increase was significant in the tumor periphery of brain and s.c. tumors. The ratio of K1s (diet to control) varied from 1.2 to 1.3 in brain tumors, 1.9 to 2.1 in subcutaneous tumors, and 1.8 to 3.1 in tumor-free brain. The apparent [3H]melphalan distribution space was significantly higher in the tumor periphery of both brain and subcutaneous tumors of the 15- and 30-min diet group. We also measured blood-brain barrier transport of [alpha-14C]aminoisobutyric acid and blood flow (with [131I]iodoantipyrine): the K1 of [alpha-14C]aminoisobutyric acid was 28.1 +/- 6.6 (SE) in brain tumors and 24.3 +/- 8.9 microliters/g/min in subcutaneous tumors. Blood flow was 58.2 --> 3.9 in brain tumors and 5.2 +/- 0.4 ml/100 g/min in subcutaneous tumors. Fasting, when combined with a protein-free diet, reduces plasma amino acid levels and thereby reduces competition between melphalan and LNAAs. This may increase the amount of melphalan that can enter a brain tumor without increasing the administered drug dose and suggests a therapeutic manipulation that can be used to increase the delivery of melphalan.

Amino Acid Transport Systems↗
PubMed1992

Radioactive assay of 2,4-dienoyl-coenzyme A reductase.

A radioactive method for assaying 2,4-dienoyl-CoA reductase, also referred to as 4-enoyl-CoA reductase (EC 1.3.1.34), is described. The assay measures the incorporation of tritium from [4B-3H]NADPH into 2-trans,4-cis-decadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA which, after cleavage of the thioester bond with hydroxylamine, can be separated from the radioactive coenzyme by extraction with toluene. This assay is at least 30 times more sensitive than the spectrophotometric assay, even though rates determined by the radioactive method are 10 times lower than rates obtained spectrophotometrically due to a primary kinetic isotope effect. The linearity of this assay with respect to time and protein concentration is sufficient for determining 2,4-dienoyl-CoA reductase activities in extracts from small samples of human fibroblasts, which were found to contain reductase activities between 1.8 and 5.8 mU/mg of protein.

Animals↗
PubMed1992

Maternal and fetal tissue distribution of L-carnitine in pregnant mice: low accumulation in the brain.

The distribution of L-carnitine was studied by whole-body autoradiography in pregnant CD-1 mice at 1, 2, 3, and 6 hr after receiving L-[14C]carnitine. Highest concentrations of carnitine were found in maternal tissues including liver, placenta, kidney, myocardium, and choroid plexus. High retention of tissue carnitine in excess of blood levels suggests the existence of concentrative uptake mechanism. Labeled carnitine was not detectable in either maternal or fetal brain. This suggests that the brain barrier systems limit the access of L-carnitine to the brain. In fetus, the level of carnitine was less than that seen in the maternal tissues, however, the tissue distribution was similar. The fetal tissue carnitine concentration increased with time. These findings suggest that relief of encephalopathy due to toxic organic anions in metabolic disorders following L-carnitine supplementation appears to be peripheral metabolic effects rather than direct access to the central nervous system. However, the physiological role for the concentrative uptake of L-carnitine by the choroid plexus remains to be determined. Transport of carnitine into fetal tissues via placenta further suggests the possibility of prenatal therapy in pregnancies at risk for certain inherited metabolic disorders.

Animals↗
PubMed1992

Octanoic acid produces accumulation of monoamine acidic metabolites in the brain: interaction with organic anion transport at the choroid plexus.

Effects of octanoic acid on monoamines and their acidic metabolites in the rat brain were analyzed by HPLC. Octanoic acid (1,000 mg/kg i.p.) elevated homovanillic acid levels by 54% in the caudate and 338% in the hypothalamus but increased 5-hydroxyindoleacetic acid (5-HIAA) levels in both the caudate and the hypothalamus by approximately 50% compared with the control. A lower dose of octanoic acid (500 mg/kg) increased 5-HIAA levels by 29% in the caudate and 20% in the hypothalamus. However, it did not produce any changes in the concentration of homovanillic acid in either the caudate or the hypothalamus. Treatment with octanoic acid also failed to change the level of dopamine, serotonin, and 3,4-dihydroxyphenylacetic acid in the caudate and the hypothalamus. The role of carrier-mediated transport in the clearance of 5-HIAA from the rabbit CSF was also evaluated in vivo by ventriculocisternal perfusion. Steady-state clearance of 5-HIAA from CSF exceeded that of inulin and was reduced in the presence of octanoic acid. Because this transport system in the choroid plexus is normally responsible for the excretion of the serotonin metabolite from the brain to the plasma, accumulation of endogenously produced organic acids in the brain, secondary to reduced clearance by the choroid plexus, could be a contributing factor in the development of encephalopathy in children with medium-chain acyl-CoA dehydrogenase deficiency who have elevated levels of octanoic acid systematically.

Acids↗
PubMed1992

Neuropsychiatric manifestations of defect in mitochondrial beta oxidation response to riboflavin.

A 29 year old woman is described with severe hyperemesis gravidarum, atypical migraine, numerous admissions to hospital for psychiatric illness, non-epileptic seizures, and valproate-induced coma. Metabolic studies and measurement of [9,10(n)-3H]palmitate oxidation by cultured fibroblasts suggested a multiple acyl-CoA dehydrogenation disorder. Treatment with riboflavin abolished headaches and abnormal behaviour and normalised the plasma free carnitine level. Subtle defects in mitochondrial beta oxidation may be a treatable cause of disordered behaviour in adults.

Acids↗
PubMed1992

Identification of a new mutation in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

A mutation involving an A-to-G nucleotide replacement at position 985 of the medium-chain acyl-CoA dehydrogenase (MCAD) cDNA was found in homozygous form in 18 unrelated MCAD-deficient families and in heterozygous form in 4 families. By PCR amplification and sequencing of cDNA from a compound heterozygote, we have detected a new mutation in an MCAD-deficient patient in whom one MCAD allele produces mRNA that is missing 4 bp in the MCAD cDNA, while the other allele carries the A-to-G-985 mutation. The presence of this 4-bp deletion was confirmed in the patient's genomic DNA by dot-blot hybridization with allele-specific oligonucleotide probes and by restriction analysis of PCR products. A rapid screening test for this 4-bp deletion was developed, based on mismatched primer PCR amplification. The deletion created a new restrictive-enzyme site which yielded two DNA fragments. The 4-bp deletion was not found in the three remaining MCAD chromosomes not harboring the A-to-G-985 mutation, nor it was present in 20 chromosomes from 10 unrelated normal Caucasians. The PCR-based method for screening these two mutations can detect over 93% of all MCAD mutations.

Acyl-CoA Dehydrogenases↗
PubMed1991

Kinetic analysis of L-carnitine uptake by the choroid plexus.

Choroid plexuses from the lateral (LVCP) and fourth ventricles (FVCP) of rats or rabbits were incubated in artificial cerebrospinal fluid (CSF) containing 1 microM [14C]L-carnitine with various concentrations of L-carnitine ranging from 0.01 mM to 1.0 mM. The time course of 1 microM [14C]L-carnitine uptake by the choroid plexus indicated that it increased linearly for the first 15 min. Steady-state levels were reached by 30 min with tissue concentrations more than 20 (FVCP)- to 30 (LVCP)-fold greater than the concentration in the medium. The uptake of [14C]L-carnitine was increased with increasing concentrations of the substrate in the medium and this uptake followed Michaelis-Menten kinetics. The uptake by the rat choroid plexus took place against a concentration gradient via a saturable process and kinetic analysis revealed Km of 32 microM (LVCP) and 34 microM (FVCP) and Vmax of 21 (LVCP) and 17 nmol/ml/min (FVCP), respectively. Ouabain inhibited the uptake by 51% (FVCP) and 48% (LVCP) and hypothermia (0 degrees C) produced inhibition by 97% (FVCP) and 96% (LVCP), respectively. However, the uptake of L-carnitine was not sensitive to probenecid or tyrosine, which indicates the presence of an independent carrier for L-carnitine in the choroid plexus. Similar results were obtained with the rabbit choroid plexus.

Animals↗
PubMed1990

L-Carnitine prevents mitochondrial damage induced by octanoic acid in the rat choroid plexus.

Cytochrome oxidase activity was investigated histochemically in the choroid plexus epithelium. Intense staining for the enzyme was exclusively limited to the mitochondria. Rats treated with octanoic acid displayed extensive ultrastructural disruptions in the epithelial cells of the choroid plexus. Mitochondria were fewer in number and more disrupted compared to the control. The enzyme activity was greatly reduced. However, pretreatment with an equimolar dose of L-carnitine followed by octanoic acid injection produced little alteration of either ultrastructure or enzyme staining. This study suggests that L-carnitine supplementation may restore mitochondrial function of the choroid plexus subjected to toxic organic anions in metabolic disorders, and may be useful in the prevention of metabolic encephalopathy.

Animals↗
PubMed1990

Identification of a common mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is one of the most common recessively inherited metabolic diseases in man. We have studied fibroblast cultures obtained from three patients with MCAD deficiency by sequencing the entire coding region of MCAD mRNA. A single A to G nucleotide replacement which resulted in lysine329-to-glutamic acid329 substitution of the MCAD protein was identified in all cultures. Furthermore, this point mutation was present in 91% (31 of 34) of mutant MCAD alleles, indicating that the majority of cases with MCAD deficiency are caused by this type of mutation.

Acyl-CoA Dehydrogenase↗
PubMed1990

Quantitative assay of free and total carnitine using tandem mass spectrometry.

A new, specific method for isotope dilution assay of total and free carnitine in urine has been developed. The method utilizes fast atom bombardment ionization with tandem mass spectrometry and requires minimal sample preparation. It compared well with radioenzymatic assay in terms of specificity, precision and accuracy, but was much more convenient in terms of analysis time and sample throughput. The new method is also applicable to the determination of free and short-chain total carnitine in plasma.

Carnitine↗
PubMed1990

Analysis of abnormal urinary metabolites in the newborn period in medium-chain acyl-CoA dehydrogenase deficiency.

In order to determine which are useful early diagnostic markers for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we have analysed urine from an asymptomatic neonate. Profiling of urinary organic acids followed by peak confirmation by electron impact mass spectrometry revealed a high suberate/adipate ratio (greater than 1.0) and the presence of n-hexanoylglycine (HG). Acylcarnitine analysis by fast atom bombardment mass spectrometry (FAB-MS) was inconclusive, but FAB-MS/MS (tandem mass spectrometry) revealed diagnostic amounts of octanoylcarnitine and hexanoylcarnitine. Quantitative analysis of acylglycines by stable isotope dilution and chemical ionization mass spectrometry revealed a 30-fold increase in HG and increased suberylglycine, but no increase in 3-phenylpropionylglycine.

Acyl-CoA Dehydrogenase↗