Kiyoshi Kidouchi

Population and family studies of dihydropyrimidinuria: Prevalence, inheritance mode, and risk of fluorouracil toxicity

To evaluate the prevalence of dihydropyrimidinuria (DHPuria), we analyzed urine samples from 21,200 healthy Japanese infants, and found two cases of DHPuria without clinical symptoms. Based on this result, we estimated the prevalence to be approximately 1/10,000 births in Japan. In addition, we analyzed pyrimidine catabolism on a previously reported family with an adult DHPuria case. We newly identified the sister of the propositus as the second case of DHPuria in this family, because she excreted large amounts of dihydrouracil and dihydrothymine. The parents and the child of the propositus showed slight increases of dihydrouracil and dihydrothymine. This is the first family with 2 cases of DHPuria, indicating that DHPuria is an inherited condition. To determine the inheritance of DHPuria in this family and to examine the risk of 5-fluorouracil (5-FU) toxicity, a uracil loading test was performed on the parents. Urinary dihydrouracil concentrations in the parents after the loading were several times higher than those in normal control persons, the finding being consistent with DHPuria heterozygotes. This, along with data on the propositus, his sister, and his child, indicates that DHPuria is an autosomal recessive condition. In addition, DHPuria homozygotes may have a high risk of 5-FU toxicity, while the risk is relatively low in heterozygotes.

Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography

An automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography (HPLC) with column switching is described. The system consists of a reversed-phase column, a cation-exchange column, a column switch, four sets of ultraviolet absorbance detectors, a microcomputer and other conventional equipment. As this system permits the simultaneous determination of urinary orotic acid, uracil, dihydrouracil, pseudouridine, xanthine, 2,8-dihydroxyadenine and succinyladenosine, it offers a useful method for the detection of orotic aciduria, dihydropyrimidine dehydrogenase deficiency, dihydropyrimidinuria, xanthinuria, adenine phosphoribosyltransferase deficiency and adenylosuccinase deficiency.

Dihydropyrimidinuria: the first case in Japan.

Dihydropyrimidinuria (McKusick 222748) is a recently described disorder of pyrimidine metabolism that presents neurological symptoms different in degree. Only two cases have been reported to date (Duran et al, 1991; Henderson et al, 1993). The patients with dihydropyrimidinuria excrete large amount of dihydrouracil and dihydrothymine, and moderate amount of uracil and thymine in urine. Therefore this disease is thought to be caused by a deficiency of dihydropyrimidine amidohydrolase (DHPase; EC 3.5.2.2), the second step of pyrimidine base catabolism. The first case, reported by Duran et al (1991), was hospitalized for convulsion and disturbed consciousness at the age of 8 weeks, but whose subsequent development had been normal. The second case reported by Henderson et al (1993) presented severe developmental delay. These two patients were discovered by the urinary gas chromatography mass spectrometry (GC-MS) analysis for the neurological sick children. We report here another case of dihydropyrimidinuria which is the first case in Japan and probably the third worldwide. We discovered her by using high-performance liquid chromatography (HPLC) at the mass screening program.

Urinary pyrimidine analysis in healthy newborns, infants, children, adults and patients with congenital metabolic diseases

Abstract Background: In previous reports, the reference range for urinary pyrimidine was determined on the basis of a small number of samples, with data for only a few patients being reported. In the present study, we measured urinary pyrimidine compounds in 25 healthy newborns, 33 healthy infants, 130 healthy children and 166 healthy adults. In addition, we also analyzed urinary pyrimidine compounds in various patients with abnormal pyrimidine metabolism, such as congenital pyrimidine metabolism disorders and urea cycle disorders.

Automated determination of 5-fluorouracil and its metabolite in urine by high-performance liquid chromatography with column switching.

We report a quantitative assay of 5-fluorouracil (FU) and its metabolite, 5-fluorodihydrouracil (FDHU) in human urine by used a column-switching high-performance liquid chromatographic method. The analyses were carried out using a molecular exclusion column for sample purification, and a cation-exchange column for separation. Each sample required only 40 min to analyze, and required no preparation other than filtration. Linearity was verified up to 1000 nmol/ml (r > 0.993). The recovery of FU was 96-101%; recovery of FDHU was 96-105%. The imprecision (RSD) for FU (10-100 nmol/ml) was < 1.5%, same-day (n = 5), and < 1.8%, day-to-day (n = 5). The imprecision (RSD) for FDHU (10-100 nmol/ml) was < 3.2%, same-day (n = 5), and < 4.0%, day-to-day (n = 5). The detection limits were, respectively, 0.1 nmol/ml. We measured FU and FDHU in urine of seven cancer patients after oral administration of FU. The cumulative quantity ratio of the FDHU and FU (FDHU/FU) excreted in their urine within 120 min after FU administration was a constant value in all seven patients. Based on these results, we believe that our method provides a useful tool for evaluating FU metabolism.

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric analysis of glycine conjugates and urinary isovalerylglycine in isovaleric acidemia

n-Acetylglycine, n-propionylglycine, n-butyrylglycine, isobutyrylglycine, n-valerylglycine, isovalerylglycine, heptanoylglycine, phenylacetylglycine and isovalerylglucuronide were identified based on their liquid chromatographic-atmospheric pressure chemical ionization mass spectra (LC-APCI-MS). We were able to detect the presence of urinary isovalerylglycine in two cases of isovaleric acidemia using LC-APCI-MS. Membrane-filtered urine samples were injected into the LC-APCI-MS system in the negative-ion mode without any further pretreatment, and large amounts of isovalerylglycine were detected as the [M-H]- ion. The urinary excretion of isovalerylglycine appeared to increase after L-carnitine therapy. This analytical method is quick and easy and it may be a useful tool in understanding dysfunctional conditions in isovaleric acidemia.

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5-20 microliters samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 +/- 14.4 mumol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.

Carnitine Deficiency in Inherited Organic Acid Disorders and Reye Syndrome

A large quantity of propionylcarnitine in the urine of patients with propionic acidemia and methylmalonic aciduria was demonstrated. The amount excreted depended on the administered L‐carnitine dose from 25 to 75 mg/kg/day. A high level of propionylcarnitine was also detected in the amniotic fluid of fetuses at risk of methylmalonic aciduria. Glutaric aciduria type 1 was characterized by excessive urinary excretion of glutarylcarnitine. In a neonate with glutaric aciduria type 2, several specific acylcarnitines were detected in the urine. These included isovaleryl‐, acetyl‐, isobutyryl‐, and butyrylcarnitine as major carnitine esters and glutaryl‐, and octanoylcarnitine as minor components. However, the pattern of acylcarnitines excreted changed from isovalerylcarnitine (via leucine) to isobutyrylcarnitine (via valine) during early life. In patients diagnosed as Reye syndrome, tissue carnitine deficiency was not always recognized and no decrease in the free/total carnitine ratio was found in the liver or muscle. The clinical and pathophysiological manifestations seen in these disorders are considered to relate to mitochondrial activity. Therefore, it is necessary to measure acylcarnitine fractions in the urine in order to obtain more precise information about mitochondrial function because carnitine and acylcarnitine compounds may express the metabolic state of mitochondria.

Allopurinol challenge tests performed before and after living-related donor liver transplantation in citrullinaemia

Summary: We performed allopurinol challenge tests to evaluate the metabolic state of a citrullinaemic patient who received a living-relative donor liver transplant. Before transplantation, large amounts of orotic acid and orotidine were excreted during the challenge test. Following transplantation, excretion of these compounds in response to allopurinol was normalised. The challenge test was a safe and useful method to evaluate the metabolic state of the patient.

Urinary Screening for Pyrimidine Metabolism Disorders

Pyrimidine chemotherapy agent such as 5-FU are used widely but can occasionally cause serious adverse reactions in patients with pyrimidine metabolism disorders. The screening method which entailed measuring dihydropyrimidine dehydro- genase activity (DPD) has been reported. This method is acceptable with small groups, but difficulties arise when dealing with large populations owing to the complicated procedure for measuring enzyme activity. We have studied the urinary screening method using high-performance liquid chromatograpy, which is acceptable with large groups.1 This method can diagnose not only dihydropyrimidine dehydrogenase deficiency but also dihydropyrimidinuria. Using this method, we have analyzed urinary dihydrouracil (DHU) and uracil concentrations in 167 healthy adults and 966 patients with malignancy, hypertension, cerebral infarction, etc. In order to establish the reference ranges of urinary pyrimidine, we used “log (concentration)”. Additionally, we calculated dihydrouracil/uracil ratio, which seemed to be reflected of DPD activity.