Identification of two novel mutations C79X and R235Q in the dihydropyrimidine dehydrogenase gene in a patient presenting with hematuria

A patient with hematuria was shown to have thymine-uraciluria. The dihydropyrimidine dehydrogenase (DPD) activity in peripheral blood mononuclear cells was 0.16 nmol/mg/h; controls: 9.9 +/- 2.8 nmol/mg/h. Analysis of DPYD showed that the patient was compound heterozygous for the novel mutations 237C > A (C79X) in exon 4 and 704G > A (R235Q) in exon 7. The nonsense mutation (C79X) leads to premature termination of translation and thus to a non-functional protein. Analysis of the crystal structure of pig DPD suggested that the R235Q mutation might interfere with the binding of FAD and the electron flow between the NADPH and the pyrimidine substrate site of DPD.     

A neonate with recurrent vomiting and generalized hypotonia diagnosed with a deficiency of dihydropyrimidine dehydrogenase

Deficiency of dihydropyrimidine dehydrogenase (DPD) is a rare inborn error of pyrimidine metabolism. To date, only about 50 patients are known worldwide. The clinical picture is varied and is not yet fully described. Most patients are diagnosed at the age of 1-3 years. We present a patient diagnosed 8 weeks postpartum.The female patient presented in the first 3 days after birth with agitation, choking, and vomiting. Six weeks later, the patient presented again with vomiting and insufficient weight gain. Metabolic screening of urine showed a strongly increased excretion of uracil and thymine, with no other abnormalities. This suggested a deficiency of DPD which was confirmed by enzyme analysis in peripheral blood mononucleair (PBM) cells (patient: activity <0.01 nmol/mg/h; controls: 9.9 +/- 2.8 nmol/mg/h). The patient was homozygous for the IVS14+1G>A mutation.MRI of the brain showed some cerebral atrophy; myelinization appeared normal. Many patients with DPD-deficiency suffer from convulsions and mental retardation, some show microcephaly, feeding difficulties, autism, and hypertonia. Our patient showed feeding difficulties and in the second half-year she developed slight motor retardation and generalized hypotonia. Further observation of the development of the patient may shed more light on the relationship between clinical symptoms and DPD deficiency. DPD deficiency may present in newborns with vomiting and hypotonia as the main symptoms.     

A Neonate with Recurrent Vomiting and Generalized Hypotonia Diagnosed with a Deficiency of Dihydropyrimidine Dehydrogenase

Deficiency of dihydropyrimidine dehydrogenase (DPD) is a rare inborn error of pyrimidine metabolism. To date, only about 50 patients are known worldwide. The clinical picture is varied and is not yet fully described. Most patients are diagnosed at the age of 1–3 years. We present a patient diagnosed 8 weeks postpartum.

The female patient presented in the first 3 days after birth with agitation, choking, and vomiting. Six weeks later, the patient presented again with vomiting and insufficient weight gain. Metabolic screening of urine showed a strongly increased excretion of uracil and thymine, with no other abnormalities. This suggested a deficiency of DPD which was confirmed by enzyme analysis in peripheral blood mononucleair (PBM) cells (patient: activity <0.01 nmol/mg/h; controls: 9.9 ± 2.8 nmol/mg/h). The patient was homozygous for the IVS14+1G>A mutation.

MRI of the brain showed some cerebral atrophy; myelinization appeared normal. Many patients with DPD-deficiency suffer from convulsions and mental retardation, some show microcephaly, feeding difficulties, autism, and hypertonia. Our patient showed feeding difficulties and in the second half-year she developed slight motor retardation and generalized hypotonia. Further observation of the development of the patient may shed more light on the relationship between clinical symptoms and DPD deficiency. DPD deficiency may present in newborns with vomiting and hypotonia as the main symptoms.     

Decreased thyroid hormone-stimulated oxygen consumption and glucose uptake in mononuclear blood cells from patients with autosomal dominant osteopetrosis type I

Nine patients, from four different families, with autosomal dominant osteopetrosis were investigated. They all had roentgenological type I disease, characterized by universal, symmetrical osteosclerosis and enlarged thickness of the cranial vault. All patients appeared clinically euthyroid. Thyroxine (T4) and tri-iodothyronine (T3) induced oxygen consumption and glucose uptake were studied in vitro in mononuclear blood cells from patients and control persons. Unstimulated oxygen consumption from patients and controls did not differ, and no difference in unstimulated glucose uptake was observed. The increase in T4 and T3 stimulated oxygen consumption was significantly lower in cells from patients with osteopetrosis (T4: 0.007 +/- 0.004 mumol/mg DNA per h, T3: 0.011 +/- 0.004 mumol/mg DNA per h) compared with controls (T4: 0.017 +/- 0.003 mumol/mg DNA per h, T3: 0.023 +/- -0.013 mumol/mg DNA per h; p less than 0.05, p less than 0.05). Cellular glucose uptake after T4 and T3 stimulation was significantly lower in patients (T4: 0.032 +/- 0.017 mmol/l per mg DNA per h, T3: 0.02 +/- 0.017 mmol/l per mg DNA per h) compared with controls (T4: 0.09 +/- 0.017 mmol/l per mg DNA per h, T3: 0.08 +/- 0.01 mmol/l per mg DNA per h; p less than 0.05, p less than 0.01). The reduced oxygen consumption and glucose uptake indicate thyroid hormone resistance which may be of pathogenetic importance for the development of autosomal dominant osteopetrosis type I.     

Enhancement of intracellular calcium concentration by extracellular ATP and UTP in Madin Darby Canine Kidney cells

Fura2 - fluorescence was utilized to test for the effect of extracellular nucleotides on intracellular calcium concentration of subconfluent Madin-Darby Canine Kidney (MDCK)-cells. Extracellular ATP (10 mumol/l) and UTP (10 mumol/l) lead to rapid (within seconds), sustained, and fully reversible enhancement of intracellular calcium concentration from 138 +/- 9 nmol/l (n = 27), to 1561 +/- 260 nmol/l (n = 10) and 3435 +/- 949 nmol/l (n = 5), respectively. Half maximal effects are observed at some 1 mumol/l. In the absence of extracellular calcium the effect of ATP is transient, pointing to release of intracellular calcium. The sustained effect in the presence of extracellular calcium indicates that the nucleotides in addition recruit calcium from extracellular space.     

The metabolism of cyclopentanol by Pseudomonas N.C.I.B. 9872

1. Pseudomonas N.C.I.B. 9872 grown on cyclopentanol as carbon source oxidized it at a rate of 228mul of O(2)/h per mg dry wt. and the overall consumption of 5.9mumol of O(2)/mumol of substrate. Cyclopentanone was oxidized at a similar rate with the overall consumption of 5.2mumol of O(2)mumol of substrate. Cells grown with sodium acetate as sole source of carbon were incapable of significant immediate oxidation of these two substrates. 2. Disrupted cells catalysed the oxidation of cyclopentanol to cyclopentanone by the action of an NAD(+)-linked dehydrogenase with an alkaline pH optimum. 3. A cyclopentanolinduced cyclopentanone oxygenase (specific activity 0.11mumol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mumol of NADPH and 0.9mumol of O(2) in the presence of 1mumol of cyclopentanone. NADPH oxidation did not occur under anaerobic conditions. The only detectable reaction product with 100000g supernatant was 5-hydroxyvalerate. 4. Extracts of cyclopentanol-grown cells contained a lactone hydrolase (specific activity 7.0mumol hydrolysed/min per mg of protein) that converted 5-valerolactone into 5-hydroxyvalerate. 5. Cyclopentanone oxygenase fractions obtained from a DEAE-cellulose column were almost devoid of 5-valerolactone hydrolase and catalysed the formation of 5-valerolactone in high yield from cyclopentanone in the presence of NADPH. 6. Incubation of 5-hydroxyvalerate with the 100000g supernatant, NAD(+) and NADP(+) under aerobic conditions resulted in the consumption of O(2) and the conversion of 5-hydroxyvalerate into glutarate. 7. The high activity of isocitrate lyase in cyclopentanol-grown cells suggests that the further oxidation of glutarate proceeds through as yet uncharacterized reactions to acetyl-CoA. 8. The reaction sequence for the oxidation of cyclopentanol by Pseudomonas N.C.I.B. 9872 is: cyclopentanol --> cyclopentanone --> 5-valerolactone --> 5-hydroxyvalerate --> glutarate --> --> acetyl-CoA.     

Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro.

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-delta-acetylornithine and S-methylcysteine in blood plasma

N-delta-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 +/- 0.4 mumol/l (range 0.8--0.2 mumol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 mumol/day). Human blood plasma contains 3.9 +/- 1.9 mumol/l (range 1.4--6.5 mumol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.     

Dihydropyrimidine Dehydrogenase Deficiency Caused by a Novel Genomic Deletion c.505_513del of DPYD

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine degradation pathway. In a patient presenting with convulsions, psychomotor retardation and Reye like syndrome, strongly elevated levels of uracil and thymine were detected in urine. No DPD activity could be detected in peripheral blood mononuclear cells. Analysis of the gene encoding DPD (DPYD) showed that the patient was homozygous for a novel c.505_513del (p.169_171del) mutation in exon 6 of DPYD.     

Plasma cholesteryl ester-triglyceride transfer protein. The catalytic domain is a low molecular weight proteolipid

The lipid transfer protein complex (LTC) isolated from human plasma by immunoaffinity chromatography transfers cholesteryl esters (CE), triglycerides, and phosphatidylcholine (PC) between lipoproteins in vitro. The molecular weight of this lipid transfer catalyst in sodium dodecyl sulfate-polyacrylamide gels was 65,000. When resolved on a gel filtration column by high performance liquid chromatography (HPLC), LTC was composed of fractions of high (greater than 150,000) to low (18,000) molecular weight, although sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of each fraction revealed bands at Mr 65,000 (major) and 52,000 (minor). The CE and triglyceride transfer activity of the low Mr HPLC fraction (1049 nmol of triglyceride/mg/h and 244 nmol of CE/mg/h) was significantly greater than that of the high Mr HPLC fraction (15-27 nmol of triglyceride/mg/h and 20-30 nmol of CE/mg/h). The PC transfer activity of the HPLC fractions was not determined. LTC proteins were separated by dialysis in acidified chloroform:methanol solution into dialysand and dialysate proteins. The dialysate contained a low Mr proteolipid, designated the catalytic domain Cd, which catalyzed CE and triglyceride transfer at equivalent rates (11.0 versus 9.5 mumol/mg/h, respectively). PC transfer activity was approximately 10% of these levels (1.5 mumol/mg/h). The dialysand consisted of a protein, designated the transfer protein TP, which facilitated CE (3.4 mumol/mg/h) preferentially over triglyceride and PC (1.0 mumol/mg/h) transfer, and a catalytically inactive protein, designated the heparin-binding domain Hd. We propose a model of the LTC protein (based on catalytic activities, monoclonal antibody reactivities, and heparin-binding capacities of the isolated proteins) in which both Hd (approximately 13 kDa) and Cd (approximately 3 kDa) originate from a single lipid transfer protein, TP.     

Inhibition of rat testicular 17 alpha-hydroxylase and 17,20-lyase activities by anti-androgens (flutamide, hydroxyflutamide, RU23908, cyproterone acetate) in vitro

Flutamide, hydroxyflutamide, RU23908 and cyproterone acetate (CPA) inhibited rat testicular microsomal 17 alpha-hydroxylase and 17,20-lyase activities in vitro. The Km of [3H] progesterone for 17 alpha-hydroxylase was 45 +/- 0.62 nmol/l (+/- SEM, n = 12) and the Km of [3H] 17 alpha-hydroxyprogesterone for 17,20-lyase was 192 +/- 0.42 nmol/l (+/- SEM, n = 12). The Ki values for 17 alpha-hydroxylase, determined from Lineweaver-Burk plots were 102 +/- 3.2 mumol/l (+/- SEM, n = 6), 363 +/- 3.8 mumol/l (+/- SEM, n = 6), 118 +/- 1.4 mumol/l (+/- SEM, n = 6) and 123 +/- 2.1 mumol/l (+/- SEM, n = 6) for flutamide, hydroxyflutamide, RU23908 and CPA respectively. Flutamide and CPA were mixed-type inhibitors, whereas hydroxyflutamide and RU23908 were competitive inhibitors of 17 alpha-hydroxylase activity. Ki values for 17,20-lyase were 33 +/- 3.1 mumol/l (+/- SEM, n = 6), 112 +/- 3.1 mumol/l (+/- SEM, n = 6), 69 +/- 4.4 mumol/l (+/- SEM, n = 6) and 71 +/- 3.2 mumol/l (+/- SEM, n = 6) for flutamide, hydroxyflutamide, RU23908 and CPA, respectively. Inhibition was found to be competitive in each case. Although the characteristic action of anti-androgens is at the receptor level, these results demonstrate that anti-androgens may also have inhibitory effects on androgen biosynthesis which could prove to be of clinical significance.     

Inhibition of human placental aromatase by mefloquine

Aromatase activity of human placental microsomes was inhibited competitively by the antimalarial drug, mefloquine, but not by the related drug, chloroquine. In the absence of any drug, the Km for testosterone was 47.1 +/- 2.3 nmol/l (mean +/- SD, n = 2). In the presence of chloroquine 500 mumol/l, the Km remained unchanged (47.4 +/- 1.8 nmol/l (mean +/- SD, n = 2), whereas mefloquine inhibited competitively with respect to substrate with a Ki value of 72 +/- 4.2 mumol/l (mean +/- SD, n = 2).     

Extrathyroidal physiology of monoiodotyrosine in humans

Normal serum monoiodotyrosine (MIT) levels (n = 152) were 0.69 +/- 0.20 nmol/l. There was wide variation of MIT levels in a 24-hour period without diurnal pattern, and there was no change throughout the menstrual cycle. MIT levels declined upon aging, but levels in hypo- and hyperthyroidism were not significantly different. MIT levels were detected in athyrotic patients (0.32 +/- 0.08 nmol/l). Desiccated thyroid raised the athyrotic MIT levels to the normal range, while levothyroxine did not. Diiodotyrosine (DIT) infusion caused an MIT rise which paralleled but lagged 1 h behind the DIT rise. These data suggest thyroidal as well as nonthyroidal sources of MIT, one of which is deiodination of DIT. Ingestion of 1 g MIT increased serum MIT to 10.6 +/- 1.7 mumol/l in women, and 7.1 +/- 2.3 mumol/l in men 30 min after ingestion; the serum half-life was 45 min.     

Purification and properties of NADH-specific dihydropteridine reductase from human erythrocytes

NADH-specific dihydropteridine reductase (EC 1.6.99.7) has been purified from human erythrocytes in essentially homogeneous form. The molecular weight of the enzyme was estimated to be 46,000 by Sephadex G-100 gel filtration. The enzyme reacted with antiserum against NADH-specific dihydropteridine reductase from bovine liver and formed a single immunoprecipitin line in the Ouchterlony double-diffusion system. This precipitin line completely fused with that formed between the human liver enzyme and the antiserum. With use of 5,6,7,8-tetrahydro-6-methylpterin, Km values of the erythrocyte enzyme for NADH and NADPH were determined to be 0.94 and 47 mumol/l, respectively. Vmax values were 58.7 mumol/min/mg with NADH and 6.41 mumol/min/mg with NADPH. The average activity of NADH-specific dihydropteridine reductase of 9 human blood samples from healthy males (20-25 years old) was calculated to be approximately 600 mU/g of hemoglobin, 1.8 mU per 20 microliters of blood, or 1.9 mU per 10(8) erythrocytes.     

The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.     

Ramipril inhibition of rabbit (Oryctolagus cuniculus) small intestinal brush border membrane angiotensin converting enzyme

1. Rabbit small intestinal brush border membranes possessed prominent angiotensin converting enzyme (ACE) activity. 2. Intestinal ACE was located on the lumen surface, as verified by ACE co-enrichment with brush border membrane marker enzymes. 3. Hydrolysis kinetics of rabbit intestinal ACE were comparable to the lung, utilizing the substrate (N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine; the Vmax = 543 +/- 51 mumol/min/g and Km = 0.62 +/- 0.09 mmol/l. 4. Intestinal brush border ACE activity was strongly inhibited by the antihypertensive drug Ramipril, which yielded an IC50 value of 5 nmol/l; the ACE activity remained completely inhibited during 15 days after a single dose of 10 mumol/l Ramipril.     

Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency affecting B lymphocytes, T lymphocytes, and platelets. Previous studies on lymphocytes from WAS patients have revealed that leu-kosialin (CD43), a cell-surface glycoprotein bearing approximately 90 O-linked oligosaccharide chains, shows an aberrant electrophoretic mobility. To determine whether this finding reflects a different pattern of O-linked glycosylation in WAS cells, we have compared healthy individuals and WAS patients with respect to glycosyltransferase activities in T lymphocytes, platelets, and Epstein-Barr virus (EBV)-immortalized B cell lines. Stimulation of peripheral T cells from normal individuals in vitro with anti-CD3 antibodies and interleukin-2 was associated with a 3-fold increase in UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) from 0.8 to 2.2 nmol/mg/h. In contrast, peripheral T lymphocytes from WAS patients showed an inversion of this phenotype with high core 2 GlcNAc-T activity in unstimulated cells (2.3 nmol/mg/h) and a 2-3-fold decrease in activity following stimulation. Core 2 GlcNAc-T activity was also three times higher in platelets from WAS patients than in normal platelets. Glycosyltransferase activities were measured in immortalized B cell lines established from WAS and normal subjects by infection with EBV. Core 2 GlcNAc-T was less than 0.4 nmol/mg/h in WAS EBV-B cell lines compared to 2.4 nmol/mg/h in EBV-B cell lines from healthy individuals, In contrast, CMP-SA:SA alpha 2-3Gal beta 1-3GalNAc-R (where SA represents sialyl (sialic acid to GalNAc) alpha 6-sialyltransferase II activity was 2.0 nmol/mg/h in the WAS EBV-B cell and less than .01 nmol/mg/h in EBV-B cell lines derived from normal subjects. Eleven other glycosyltransferase activities were measured and found to be similar in EBV-B cell lines from WAS and normal individuals. Polylactosamine sequences were much reduced in the O-linked oligosaccharides of CD43 from WAS EBV-B cells consistent with decreased core 2 GlcNAc-T activity and expression of core 1 oligosaccharides in the cells. In conclusion, B cells, T cells, and platelets in WAS patients show abnormal expression of two developmentally regulated glycosyltransferases, consistent with the idea that the WAS immunodeficiency is due to a failure of normal lymphocyte maturation.     

Mevalonate kinase in lysates of cultured human fibroblasts and lymphoblasts: kinetic properties, assay conditions, carrier detection and measurement of residual activity in a patient with mevalonic aciduria

An assay has been developed for the measurement of mevalonate kinase activity in extracts of cultured human fibroblasts and lymphoblasts. Individual elements of the assay were investigated in order to achieve optimum conditions. Apparent Michaelis constants (KMapp) for the substrates mevalonic acid and adenosine-5'-triphosphate were 22 +/- 10 mumol/l and 0.42-0.53 mmol/l, respectively, in lysates of control fibroblast lines. The same values in lysates of a control lymphoblast line were 17 mumol/l and 0.23 mmol/l, respectively. Mevalonate kinase activity in extracts of cultured fibroblasts derived from 6 control individuals was 3.24 +/- (SD) 0.91 nmol/min/mg protein. The activity in extracts of fibroblasts derived from a patient with mevalonic aciduria was 0.15 +/- 0.10 nmol/min/mg protein, approximately 5% of the control mean. The parents and brother of the patient displayed mevalonate kinase activities in fibroblast extracts approximating 38-42% of the control mean. Substantially higher mevalonate kinase activity was documented in extracts of cultured lymphoblasts. When assayed on various occasions, the mean activity of mevalonate kinase in extracts of lymphoblasts derived from the parents, brother and maternal grandmother of the patient ranged from 27 to 32% of the mean activity of 9.8 +/- (SD) 3.4 nmol/min/mg protein measured in a parallel control lymphoblast line, while the mean activity in a maternal and paternal uncle approximated 65-89% of the same control mean. The mean activity in extracts of lymphoblasts derived from the patient approximated 2% of the control mean. The data suggest that the parents, brother and maternal grandmother are carriers of the defective gene responsible for mevalonate kinase deficiency, consistent with an autosomal recessive mode of inheritance.     

Bile pigment formation and excretion in the rabbit

Bile and plasma levels of biliverdin and bilirubin, together with the hepatic biliverdin reductase and bilirubin UDP-glucuronosyl transferase activities, were studied in the rabbit. No biliverdin could be detected in the blood plasma. The bilirubin concentration in blood was 7.81 +/- 0.79 mumol/l. Biliverdin was the predominant pigment in bile (63%). Hepatic biliverdin reductase activity was 0.086 +/- 0.016 nmol/mg protein/hr. The synthesis of bilirubin was apparently limited by the enzyme activity. Most of the bilirubin in bile was conjugated (90%) with monoconjugates predominating (75%). Hepatic UDP-glucuronosyl transferase activity was 2.65 +/- 0.18 and 1.14 +/- 0.16 mumol/mg protein/hr with and without activation, respectively.     

Metabolism of androgens in human benign prostatic hyperplasia: aromatase and its inhibition

As an extension of our studies on androgen metabolism in epithelium and stroma of human benign prostatic hyperplasia (BPH) tissue our attempts to demonstrate the presence of aromatase are described. Additionally, the question is raised whether the aromatase inhibitor 17 alpha-oxa-D-homoandrosta-1.4-diene-3.17-dione (testolactone) might also act by inhibition of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSDH). In vitro metabolism and inhibition were analyzed by TLC. The main results were: (1) Two aromatase assays (estrone formation and tritium release) were tested with placenta microsomes. Identical results were obtained (Km = 43 +/- 7 nmol/l n = 5; Vmax = 100 resulted in recovery of the aromatase activity added. (3) In BPH tissue alone, formation of estrone from androstenedione could not be detected (less than 7 x 10(-17) mol/min per mg protein, n = 8). (4) 4-Hydroxyandrostenedione inhibited placental aromatase (Ki = 37 nmol/l) distinctly better than 17 beta-HSDH from human BPH (Ki = 18 mumol/l), whereas the Ki values for testolactone (3.7 and 29 mumol/l, respectively) were more similar. It is concluded that aromatization of androgens is not an important pathway in BPH tissue. An alternative mode of action of testolactone by inhibition of 17 beta-HSDH is discussed.