Ethacrynic acid effect on the composition of cochlear fluids.

Ethacrynic acid, when administered to six dogs, caused a fall in the concentration of potassium in endolymph from 145 to 21 milliequivalents per liter and a rise in the concentration of sodium from 5.9 to 146 milliequivalents per liter. The composition of perilymph is unaffected. Perilymph and endolymph are slightly hypertonic as compared to plasma.

Cerebrospinal fluid production: stimulation by cholera toxin.

Large increases in the production of cerebrospinal fluid have been observed after the intraventricular administration of cholera toxin. Because cholera toxin stimulates adenylate cyclase, the data suggest that adenosine 3',5'-monophosphate plays a role in cerebrospinal fluid production.

Arginine, an indispensable amino acid for patients with inborn errors of urea synthesis.

The role of arginine as an essential amino was evaluated in four children with one of the deficiencies of carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinase. Within 15-68 h after arginine deprivation nitrogen accumulated as ammonium or glutamine or both, but glutamine was quantitatively the largest nitrogen accumulation product. Concomitantly plasma and urinary urea levels decreased. Resumption of arginine intake (or citrulline in the case of ornithine transcarbamylase deficiency) promptly led to correction of the hyperammonemia, hyperglutaminemia and hypoargininemia. Ornithine was an unsatisfactory substitute for arginine. Arginine deprivation did not interfere with carbamyl phosphate synthesis as manifested by orotic aciduria. It is concluded that arginine is an indispensable amino acid for children with inborn errors of ureagenesis and its absence results in the rapid onset of symptomatic hyperammonemia.

The mutual independence of the endolymphatic potential and the concentrations of sodium and potassium in endolymph.

The relationship between the endolymphatic potential (EP) and the sodium and potassium concentration gradients between endolymph and interstitial fluid was studied both by measuring the EP at varying concentrations of sodium and potassium in endolymph and by measuring the effect of a depressed EP on the concentrations of these cations. Ethacrynic acid was used in dogs to change the concentration of sodium and potassium (meq/liter) in endolymph from 5.8 and 148 to 134 and 24.3, respectively. No change in the EP accompanied these alterations. In a second series of experiments the EP was reduced from + 72 mV to + 31 mV for a mean duration of 20 min. No change in the concentration of sodium and potassium in endolymph was found during the period of reduced EP. These data suggest that there is little relationship between the EP and the sodium and potassium concentrations in endolymph.

Gyrate atrophy of the choroid and retina: amino acid metabolism and correction of hyperornithinemia with an arginine-deficient diet.

Four patients with gyrate atrophy of the choroid and retina were studied, all of whom exhibited the hyperornithinemia characteristic of this disorder. Elevated plasma histidine and diminished plasma lysine and branched-chain amino acids were also noted. The renal clearances of these four amino acids were not sufficiently elevated to explain their low plasma levels. In one subject, an arginine-deficient diet led to progressive reduction in plasma ornithine from 13 times normal to the upper limits of normal, along with the disappearance of ornithinuria and lysinuria. Orally administered alpha-aminoisobutyric acid facilitated the fall in plasma ornithine by increasing renal losses of ornithine. It also increased the clearances of most other amino acids. When plasma ornithine approached normal (less than 200 microM), plasma lysine became normal, plasma arginine became subnormal, and renal clearances of basic amino acids decreased. Long-term (1.5 yr) maintenance with a diet containing 10-20 g of protein plus essential amino acids served to keep plasma ornithine at between 55-355 microM; chorioretinal degeneration did not progress and vision apparently improved.

Effect of glutamine synthetase inhibition on astrocyte swelling and altered astroglial protein expression during hyperammonemia in rats.

Inhibition of glutamine synthesis reduces astrocyte swelling and associated physiological abnormalities during acute ammonium acetate infusion in anesthetized rats. We tested the hypothesis that inhibition of glutamine accumulation during more prolonged ammonium acetate infusion in unanesthetized rats reduces cortical astrocyte swelling and immunohistochemical changes in astrocytic proteins. Rats received a continuous i.v. infusion of either sodium acetate or ammonium acetate for 24 h to increase plasma ammonia from about 30-400 mumol/l. Cohorts were pretreated with vehicle or l-methionine-S-sulfoximine (MSO; 0.83 mmol/kg). MSO reduced glutamine synthetase activity by 57% and glutamine synthetase immunopositive cell number by 69%, and attenuated cortical glutamine accumulation by 71%. Hyperammonemia increased the number of swollen astrocytes in cortex and MSO reduced this increase to control values. The number of glial fibrillary acidic protein immunopositive cells in cortex was greater in hyperammonemic rats and the increase in superficial cortical layers was attenuated by MSO. Immunoreactivity for the gap junction protein connexin-43 in the neuropil, assessed by optical density, was greater in the hyperammonemic group compared with controls, but this increase was not attenuated by MSO. No changes in the optical density of GLT1 glutamate transporter immunoreactivity in cortex were detected in any group. We conclude that glutamine synthetase inhibition reduces astrocyte swelling and ameliorates some of the reactive astroglial cytoskeletal alterations seen at 24 h of hyperammonemia, but that gap junction changes in astrocytes occur independently of glutamine accumulation and swelling.

Preservation of cerebral blood flow responses to hypoxia and arterial pressure alterations in hyperammonemic rats.

Acute hyperammonemia causes cerebral edema, elevated intracranial pressure and loss of cerebral blood flow (CBF) responsivity to CO2. Inhibition of glutamine synthetase prevents these abnormalities. If the loss of CO2 responsivity is secondary to the mechanical effects of edema, one would anticipate loss of responsivity to other physiological stimuli, such as hypoxia and changes in mean arterial blood pressure (MABP). To test this possibility, pentobarbital-anesthetized rats were subjected to either hypoxic hypoxia (PaO2 approximately 30 mm Hg), hemorrhagic hypotension (MABP approximately 70 and 50 mm Hg), or phenylephrine-induced hypertension (MABP approximately 125 and 145 mm Hg). CBF was measured with radiolabeled microspheres. Experimental groups received intravenous ammonium acetate (approximately 50 mumol min-1 kg-1) for 6 h to increase plasma ammonia to 500-600 microM. Control groups received sodium acetate plus HCl to prevent metabolic alkalosis. The increase in CBF during 10 min of hypoxia after 6 h of ammonium acetate infusion (84 +/- 19 to 259 +/- 52 ml min-1 100 g-1) was similar to that after sodium acetate infusion (105 +/- 20 to 265 +/- 76 ml min-1 100 g-1). Cortical glutamine concentration was elevated equivalently in hyperammonemic rats subjected to normoxia only or to 10 min of hypoxia. With severe hypotension, CBF was unchanged in both the ammonium (80 +/- 20 to 76 +/- 24 ml min-1 100 g-1) and the sodium (80 +/- 14 to 73 +/- 16 ml min-1 100 g-1) acetate groups. With moderate hypertension, CBF was unchanged. With the most severe hypertension, significant increases in CBF occurred in both groups, but there was no difference between groups. We conclude that hypoxic and autoregulatory responses are intact during acute hyperammonemia. The previously observed loss of CO2 responsivity is not the result of a generalized vasoparalysis to all physiological stimuli.

Methionine sulfoximine, a glutamine synthetase inhibitor, attenuates increased extracellular potassium activity during acute hyperammonemia.

Hyperammonemia causes glutamine accumulation and astrocyte swelling. Inhibition of glutamine synthesis reduces ammonia-induced edema formation and watery swelling in astrocyte processes. Ordinarily, astrocytes tightly control extracellular K+ activity [K+]e. We tested the hypothesis that acute hyperammonemia interferes with this tight regulation such that [K+]e increases and that inhibition of glutamine synthetase reduces this increase in [K+]e. Ion-sensitive microelectrodes were used to measure [K+]e in parietal cortex continuously over a 6-h period in anesthetized rats. After i.v. sodium acetate infusion in eight control rats, plasma ammonia concentration was 33 +/- 26 mumol/L (+/- SD) and [K+]e remained stable at 4.3 +/- 1.6 mmol/L. During ammonium acetate infusion in nine rats, plasma ammonia increased to 594 +/- 124 mumol/L at 2 h and to 628 +/- 135 mumol/L at 6 h. There was a gradual increase in [K+]e from 3.9 +/- 0.7 to 6.8 +/- 2.7 mmol/L at 2 h and 11.8 +/- 6.7 mmol/L at 6 h. In eight rats, L-methionine-D,L-sulfoximine (150 mg/kg) was infused 3 h before ammonium acetate infusion to inhibit glutamine synthetase. At 2 and 6 h of ammonium acetate infusion, plasma ammonia concentration was 727 +/- 228 and 845 +/- 326 mumol/L, and [K+]e was 4.5 +/- 1.9 and 6.1 +/- 3.8 mmol/L, respectively. The [K+]e value at 6 h was significantly less than that obtained with ammonium acetate infusion alone but was not different from that obtained with sodium acetate infusion. We conclude that acute hyperammonemia impairs astrocytic control of [K+]e and that this impairment is linked to glutamine accumulation rather than ammonium ions per se.

The phenotype of ostensibly healthy women who are carriers for ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder of urea synthesis. Among females who carry a mutant OTC allele, there is a wide range of phenotypic variability, ranging from apparent normality to a severe onset and the resulting profound neurologic impairment observed in hemizygous males. This study was designed to define the phenotypic variability of OTC deficiency in ostensibly healthy carrier females and to compare them to noncarrier females from their own and other families. One hundred seventy-five women from 89 families participated in this study. Each completed a mailed questionnaire, allopurinol testing, and fasting plasma amino acid determinations. OTC carrier status was determined by pedigree analysis, allopurinol test results, and/or DNA mutation analysis. Overall, 79 women were identified as carriers of a mutant OTC allele (60 proband mothers, 19 relatives), and 96 women (32 proband mothers, 64 female relatives) were determined to be noncarriers. Comparison of biochemical phenotypes indicated that carriers and noncarriers do not differ in daily urinary creatinine excretion, but that carriers excrete significantly less urea nitrogen and total nitrogen, reflecting their significantly lower historically reported daily protein intake. Carriers had significantly higher levels of fasting plasma glutamine and alanine, and significantly lower levels of citrulline and arginine compared with noncarriers. Carriers and noncarriers reported similar demographic characteristics, anthropometric measurements, level of education, and medical and pregnancy histories. There was no indication of increased incidence of migraine headaches among carriers. Thus, we found no evidence that asymptomatic adult female OTC heterozygotes are at increased risk for previously unidentified health problems apart from an unknown risk for hyperammonemic encephalopathy as occurred in 3 of the carriers in this study. Because these episodes appear to be related to physiologic stress (fracture, parturition), it would seem medically prudent for carriers to be aware of this risk.

Syndrome of idiopathic hyperammonemia after high-dose chemotherapy: review of nine cases.

PURPOSE: The syndrome of idiopathic hyperammonemia occurs in patients who have received high-dose cytoreductive therapy for the treatment of hematologic malignancy. It is characterized by abrupt alteration in mental status and respiratory alkalosis associated with markedly elevated plasma ammonium levels in the absence of any identifiable cause, and frequently results in intractable coma and death. Our goal was to survey clinical and pathologic manifestations of the disorder and discuss treatment options. PATIENTS AND METHODS: Plasma ammonium levels were measured in patients on the acute leukemia service or on the bone marrow transplant service at The Johns Hopkins Hospital, and a level more than twice normal was considered diagnostic of hyperammonemia. The syndrome was identified in nine patients; in eight, hyperammonemia occurred after administration of intensive cytoreductive therapy that resulted in profound leukopenia. The disorder occurred in the ninth patient two months after allogeneic bone marrow transplantation. RESULTS: Three of the nine patients survived an episode of idiopathic hyperammonemia; one patient subsequently died of leukemia and one of recurrent idiopathic hyperammonemia. The one long-term survivor is currently alive and well without neurologic sequelae 250 days after autologous bone marrow transplantation. CONCLUSION: Because neurologic function can deteriorate rapidly, early recognition of this disorder and close monitoring of the patient's neurologic status are critical.

Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat.

Astrocyte hypertrophy and swelling occur in a variety of pathophysiological conditions, including diseases associated with hyperammonemia. Ammonia is rapidly incorporated into glutamine by glutamine synthetase localized in astrocytes. We tested the hypotheses that (1) 6 h of hyperammonemia (500-600 microM) is adequate for producing astrocyte enlargement, and (2) astrocyte enlargement is attenuated by inhibition of glutamine synthetase with methionine sulfoximine. Pentobarbital-anesthetized rats received an intravenous infusion of either sodium or ammonium acetate after intraperitoneal pretreatment with vehicle, methionine sulfoximine (0.8 mmol/kg) or buthionine sulfoximine (4 mmol/kg), an analogue that does not inhibit glutamine synthetase. Hyperammonemia produced enlarged cortical astrocytes characterized by (1) decreased electron density of cytoplasmic matrix in perikaryon, processes and perivascular endfeet, (2) increased circumference of nuclear membrane, (3) increased numbers of mitochondria and rough and smooth endoplasmic reticulum in perikarya and large processes, and (4) less compact bundles of intermediate filaments. Pretreatment with methionine sulfoximine, but not buthionine sulfoximine, attenuated the decrease in cytoplasmic density and the increase in nuclear circumference; most perivascular endfeet remained as dense as occurred with sodium acetate infusion. However, increased numbers of organelles in expanded perikarya and large processes occurred after methionine sulfoximine treatment with and without ammonium acetate infusion. In separate groups of rats, hyperammonemia produced an increase in cortical tissue water content which was inhibited by methionine sulfoximine, but not buthionine sulfoximine. We conclude that clinically-relevant levels of hyperammonemia can cause astrocyte enlargement within 6 h in vivo characterized by both watery cytoplasm and increased organelles indicative of a cellular metabolic stress and altered astrocyte function. The watery cytoplasm component of astrocyte enlargement depends on glutamine synthesis rather than on ammonium ions per se, and is possibly caused by the osmotic effect accumulated glutamine.

Percutaneous transluminal angioplasty of homograft renal artery stenosis in a 10-year-old girl.

Hypertension caused by 90% stenosis of a transplant renal artery was effectively treated by percutaneous transluminal angioplasty of the stenosed artery. Following dilation no pressure gradient existed across the stenosis. Hypertension had developed in a 10-year-old girl with caudal regression syndrome and recurrent pylonephritis five months following homograft transplantation for chronic renal failure. Since percutaneous transluminal angioplasty was performed, the blood pressure has remained within age-related normal limits.

First report of management and outcome of pregnancies associated with hereditary orotic aciduria.

Two pregnancies in a 25-year-old woman with hereditary orotic aciduria who was managed prenatally on uridine therapy are described. The first pregnancy resulted in an infant with multiple congenital anomalies and a 47,xx,inv(4)(p12q25), +der(22)t(11;22)(p23;q11) karyotype. The proposita was found to be a carrier of a de novo 11;22 translocation and a pericentric inversion of chromosome 4. Subsequently, several carriers of orotic aciduria in this family were identified with the inverted chromosome 4. The second pregnancy resulted in a normal male with an inverted chromosome 4.

Restoration of nitrogen homeostasis in a man with ornithine transcarbamylase deficiency.

We evaluated the hypothesis that sodium phenylbutyrate-induced phenylacetylglutamine biosynthesis in a man with partial ornithine transcarbamylase (OTC) deficiency has a dual effect; it provides an additional vehicle for waste nitrogen excretion, and in the process it suppresses the patient's residual urea N synthesis, which then may be available for N homeostasis if the need arises. A 38-year-old man was studied over three periods. Period I was a control period during which he received a fixed caloric and N intake plus L-citrulline. Phenylbutyrate was added in period II and was maintained during period III, during which his N intake was increased. Plasma levels of ammonium and glutamine and net urea N synthesis were measured in each period; phenylacetylglutamine N synthesis was measured in periods II and III. These studies demonstrated that phenylbutyrate administration led to a 73% decrease in net de novo urea N synthesis during period II, which subsequently increased threefold in period III in response to the increased N intake. Phenylacetylglutamine N synthesis was 2.27 g/d, similar to his estimated maximum net urea N synthesis of 2.65 g/d. During periods II and III, his plasma levels of ammonium and glutamine improved as compared with period I when they were abnormally high. We conclude that sodium phenylbutyrate treatment of patients with urea cycle disorders who have significant residual enzyme activity results in both an improvement in waste N excretion and improved N homeostasis as a result of the generation of a reserve urea N synthetic capacity. This therapeutic approach may be useful in other nitrogen accumulation decreases, eg, portal-systemic encephalopathy.

Effect of cholera toxin and prostaglandins on the rat choroid plexus in vitro.

The present study was performed to measure the effects of cholera toxin (CT) and prostaglandins (PG) on choroid plexus cyclic AMP accumulation. Choroid plexuses, isolated from Sprague-Dawley rats, were incubated in Krebs-Ringer bicarbonate buffer containing either CT (10 microgram/ml), heat inactivated CT, prostaglandins, or appropriate controls. After a minimum incubation period of 60 min with CT, cyclic AMP accumulation (pmol/mg protein) in treated incubates (58.10 +/- 6.43, n = 6) was 4 times that in controls (14.-5 +/- 1.4, n = 6). This large increase was only seen when theophylline (10 mM) was in the incubation mixture 3--10 min prior to the end of the incubation period. The increase in cyclic AMP accumulation was dose responsive and was irreversible after 5 min of incubation. Of the 5 prostaglandins studied, only PGE2 resulted in an increase in cyclic AMP accumulation. Cyclic AMP levels were 29.64 +/- 2.5 in controls and 57.57 +/- 3.5 in plexus which had been incubated with PGE2 (20 microgram/ml) for 1 min, and this increase was dose responsive.

The effect of cholera toxin on choroid plexus carbonic anhydrase activity in vitro.

The effects of the adenylate cyclase agonists cholera toxin and prostaglandin E2 on carbonic anhydrase activity in vitro was measured in choroid plexuses isolated from Sprague-Dawley rats. Choroid plexuses were incubated in buffer at 38 degrees C (pH 7.4) with either cholera toxin or prostaglandin E2 (PGE2) at a concentration and for a time period that had been shown in earlier studies to result in maximal stimulation of cyclic AMP production. Cholera toxin (10 micrograms/ml) caused a twofold increase (p < .001) in choroid plexus carbonic anhydrase activity when cholera toxin treated plexuses [20.92 +/- .46 mol CO2/(min)(mg protein X 10(-8)] were compared with plexuses exposed to heat inactivated cholera toxin (10.92 +/- .43). When choroid plexuses were homogenized and separated into a 10,000 g pellet and a supernatant fraction, the supernatant carbonic anhydrase was unresponsive to cholera toxin stimulation. In the pellet fraction, which contained all the cellular adenylate cyclase, challenge with cholera toxin produced a significant increase in carbonic anhydrase activity (p < .01). Control activity was 10.9 +/- 1.2 mol CO2/(min)(mg protein X 10(-8), while carbonic anhydrase activity in fractions exposed to cholera toxin was 28.4 +/- 0.8. PGE2 had no effect, however, upon choroid plexus carbonic anhydrase activity. Since both PGE2 and cholera toxin stimulate cyclic AMP production in vitro, a compartmental model of secretory control is proposed.

Measurement of the hydrostatic pressures of the cochlear compartments.

A micropressure transducer (sensitive 0.1 cm H2O) utilizing a manual servo nulling system and a micropipette (tip diameter 12-20 micron) was designed in order to measure endolymphatic and perilymphatic hydrostatic pressures in the inner ear of the guinea pig. Perilymphatic pressures were measured through the round window membrane in animals in which the ossicular structures had been removed and in those in which the middle ear structures were intact. Endolymphatic pressures were measured after removal of the middle ear structures. There was a significant (p less than 0.001) difference between perilymphatic pressures in the presence (4.7 +/- 0.36 cm H2O) and absence (2.43 +/- 0.22 cm H2O) of the middle ear structures. The endolymphatic pressure was 0.00 cm H2O when measured through the basilar membrane after disruption of the scala tympani, and was 3.34 +/- 0.57 when monitored through the spiral ligament and stria vascularis. In order to verify the accuracy of these measurements, we monitored pressures in animals whose perilymphatic pressures were artificially maintained by an external source. Recoveries were always 95-100% of the artificially applied pressure. The injection of purified cholera toxin into the scala media through the basilar membrane resulted in a significant (p less than 0.001) increase in endolymphatic pressure.

Allopurinol-induced pyrimidinuria in cancer patients.

Allopurinol induced pyrimidinuria is a sensitive and specific test that identifies the increased de novo pyrimidine mononucleotide biosynthesis accompanying ornithine trans carbamylase deficiency. We hypothesize that the increased de novo DNA synthesis characteristic of malignant tumors can be detected using this method. Eleven cancer patients and a 30 subject control group were studied. The allopurinol test protocol consists of five urine collection periods--a baseline collection after which time a 300 mg dose of allopurinol is taken, followed by collection of four 6 hour fractional urine collections. Orotate, orotidine and creatinine were measured on the samples. Eight of 11 patients had significantly elevated urine levels of either orotate or orotidine either prior to challenge with allopurinol or during one of the time periods. The allopurinol test for this unstratified group has a sensitivity of 0.73 and a specificity of 1.0. This observational and preliminary report suggest that further study of the potential significance and usefulness of the allopurinol test in patients at risk for or with malignancy is warranted.