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.

Three cases of intravenous sodium benzoate and sodium phenylacetate toxicity occurring in the treatment of acute hyperammonaemia.

Intravenous sodium benzoate and sodium phenylacetate have been used successfully in the treatment of acute hyperammonaemia in patients with urea cycle disorders. They provide alternative pathways for waste nitrogen disposal and help maintain nitrogen homeostasis. However, we report three patients with hyperammonaemia who received inappropriate doses of intravenous sodium benzoate and sodium phenylacetate that resulted in severe complications. Ambiguous medical prescriptions and inadequate cross-checking of drug dosage by physicians, nurses and pharmacists were the main causes of these incidents. All the patients presented with alteration in mental status, Kussmaul respiration and a partially compensated metabolic acidosis with an increased anion gap. Two patients developed cerebral oedema and hypotension and died. The third survived after haemodialysis. Plasma levels of benzoate and phenylacetate were excessively high. The possible mechanisms of toxicity, management and safety measures are discussed.

Interaction of glutamine and arginine on cerebrovascular reactivity to hypercapnia.

Glutamine is purported to inhibit recycling of citrulline to arginine and to limit nitric oxide release in vitro. However, vasoactive effects of glutamine have not been clearly demonstrated in vivo. During hyperammonemia, impaired cerebrovascular reactivity to CO(2) is related to glutamine accumulation. We tested the hypotheses that 1) glutamine infusion in the absence of hyperammonemia impairs cerebrovascular CO(2) reactivity and 2) arginine infusion preserves CO(2) reactivity during glutamine infusion and during hyperammonemia. Pentobarbital sodium-anesthetized rats were equipped with a closed cranial window for measuring pial arteriolar diameter. Intravenous infusion of 3 mmol. kg(-1). h(-1) of L-glutamine for 6 h produced threefold increases in plasma and cerebrospinal fluid concentrations. Dilation to hypercapnia was reduced by 45% compared with that of a time control group at 6 h but not at 3 h of glutamine infusion. Coinfusion of 2 mmol. kg(-1). h(-1) of L-arginine with glutamine maintained the hypercapnic vasodilation at the control value. Infusion of ammonium acetate at a rate known to produce threefold increases in cortical tissue glutamine concentration resulted in no significant hypercapnic vasodilation. Coinfusion of arginine with ammonium acetate maintained hypercapnic vasodilation at 60% of the control value. Arginine infusion did not augment hypercapnic vasodilation in a control group. We conclude that glutamine modulates cerebrovascular CO(2) reactivity in vivo. Glutamine probably acts by limiting arginine availability because the vascular inhibitory effect required >3 h to develop and because arginine infusion counteracted the vascular effect of both endogenously and exogenously produced increases in glutamine.

Neonatal onset ornithine transcarbamylase deficiency: A retrospective analysis.

OBJECTIVE: A retrospective analysis of 74 cases of neonatal-onset ornithine transcarbamylase (OTC) deficiency. METHODS: The medical records of 74 of the 128 male patients referred to this center with neonatal onset OTC from 1976 to 1996 were available and analyzed. RESULTS: Initial symptoms of OTC deficiency were nonspecific and included feeding difficulties, lethargy, and "respiratory distress"; vomiting was infrequent. Respiratory alkalosis was regularly observed; the mean pH and pCO2 were 7.5 and 24 torr, respectively. Early consideration of a metabolic disorder in those neonates with a negative family history was only 9%. Sepsis was initially misdiagnosed in 50% of the cases. For all patients the mean age at onset was 63 hours. Survival was better among those who had later onset, later diagnostic studies, and diagnosis. Apart from 1 patient whose peak ammonium level was 400 micromol/L, all surviving patients had severe developmental delay. CONCLUSIONS: OTC deficiency should be suspected in term infants who have early signs of encephalopathy, particularly after the first 24 hours; a respiratory alkalosis is pathognomic of urea cycle disorders. Severe developmental delay is the usual outcome of OTC deficiency.

Preserved hypocapnic pial arteriolar constriction during hyperammonemia by glutamine synthetase inhibition.

Ammonia intoxication, which results in astrocytic edema and glutamine accumulation, blocks cerebral vasodilation during hypercapnia but not during hypoxia. Ammonia's effect on blood flow during hypocapnia is unclear, with some brain regions showing a paradoxical increase in flow. Here, we studied the responses to hypocapnia of pial arterioles not surrounded by astrocytic end feet to avoid mechanical compression by local edema. Blood flow was measured by microspheres in pentobarbital sodium-anesthetized rats equipped with closed cranial windows that permitted intravital microscopy. The normal pial arterial constriction in hypocapnia (12 +/- 1%; mean +/- SE) was blocked (2 +/- 1%) during a 6-h intravenous infusion of ammonium acetate, with some regions (cerebrum, midbrain) showing increased flow during hypocapnia. After pretreatment with methionine sulfoximine (MSO), which inhibits glutamine synthesis, the normal hypocapnic constrictor response was retained in pial arterioles (11 +/- 2%) during hyperammonemia. The increase in the calculated cerebrovascular resistance also was retained. An analog of MSO that does not block glutamine synthesis (buthionine sulfoximine) was ineffective in maintaining hypocapnic reactivity. In a sodium acetate-treated control group, MSO did not alter the pial arteriolar response. Normal vasoconstrictive ability was shown during ammonium infusion in response to U-46619, a thromboxane analog. We conclude that the inhibition of hypocapnic responsivity induced by ammonium is not due to paralysis of the pial arteriolar smooth muscle or to vascular compression by swollen astrocytes but is in some way due to glutamine metabolically produced from the ammonium.

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.

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.

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.

Long-term treatment of girls with ornithine transcarbamylase deficiency.

BACKGROUND: Ornithine transcarbamylase is an X-linked mitochondrial enzyme that catalyzes the synthesis of citrulline from carbamoyl phosphate and ornithine. A deficiency of this enzyme leads to hyperammonemia and hyperglutaminemia. In boys the disease is often fatal when its onset occurs during the neonatal period, but it is milder when onset occurs later in childhood. Heterozygous girls may be normal or may have episodes of hyperammonemic encephalopathy and decline in cognitive function. We report here on the long-term outcome in girls with ornithine transcarbamylase deficiency enrolled in studies of treatments designed to activate new pathways of waste-nitrogen excretion. METHODS: We studied 32 girls (age, 1 to 17 years) with ornithine transcarbamylase deficiency who had had at least one episode of encephalopathy. The patients were assigned to treatment that consisted of sodium benzoate, alone or in combination with sodium phenylacetate or sodium phenylbutyrate, or sodium phenylbutyrate alone. Collaborating physicians provided clinical, metabolic, and developmental data at specified intervals. RESULTS: Patients treated according to these protocols had greater than 90 percent survival at five years and maintained appropriate weight for height. The frequency of hyperammonemic episodes decreased with increasing age and with sodium phenylacetate or sodium phenylbutyrate treatment. Although the mean IQ before treatment was in the low average range, 19 of the 23 girls in whom intelligence was tested longitudinally had stable test scores. CONCLUSIONS: Girls with symptomatic ornithine transcarbamylase deficiency who are treated with drugs that activate new pathways of waste-nitrogen excretion have fewer hyperammonemic episodes and a reduced risk of further cognitive decline.

Impaired pial arteriolar reactivity to hypercapnia during hyperammonemia depends on glutamine synthesis.

BACKGROUND AND PURPOSE: Acute hyperammonemia causes glutamine and water accumulation in astrocytes and loss of the cerebral blood flow response selectively to CO2. We tested whether extraparenchymal pial arterioles not subjected directly to mechanical compression by swollen astrocyte processes also lose hypercapnic reactivity and whether any such loss can be attenuated by inhibiting glutamine synthesis during hyperammonemia. METHODS: Pentobarbital-anesthetized rats were pretreated intravenously with either saline vehicle, methionine sulfoximine (0.83 mmol/kg), which inhibits glutamine synthetase and potentially gamma-glutamylcysteine synthetase, or buthionine sulfoximine (4 mmol/kg), which inhibits gamma-glutamylcysteine synthetase. Three hours after pretreatment, cohorts received an intravenous infusion of either sodium or ammonium acetate for 6 hours. Pial arteriolar diameter was measured with radiolabeled microspheres during normocapnia and 10 minutes of hypercapnia. RESULTS: With sodium acetate infusion, pial arteriolar diameter increased during hypercapnia in groups pretreated with vehicle (23+/-3% [mean+/-SE]; n=6), methionine sulfoximine (37+/-11%; n=5), and buthionine sulfoximine (32+/-3%; n=5). With ammonium acetate infusion, pial arteriolar diameter increased only in the group pretreated with methionine sulfoximine (31+/-4%; n=8) but not in those pretreated with vehicle (-2+/-4%; n=8) or buthionine sulfoximine (4+/-4%; n=6). Methionine sulfoximine, but not buthionine sulfoximine, also prevented loss of the cerebral blood flow response to hypercapnia, an increase in cortical tissue water content, and an increase in pressure under the cranial window during normocapnia in hyperammonemic rats. In contrast to hypercapnia, hypoxemia increased arteriolar diameter 30+/-7% (n=5) during ammonium acetate infusion. CONCLUSIONS: Loss of the blood flow response to hypercapnia during acute hyperammonemia is not due simply to swollen astrocyte processes passively impeding blood flow because extraparenchymal resistance arterioles also lose their reactivity selectively to hypercapnia. Lost reactivity depends on glutamine synthesis rather than on ammonium ions per se and may reflect indirect effects of astrocyte dysfunction associated with glutamine accumulation or possibly effects of glutamine on nitric oxide production.

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.

Long-term survival of patients with argininosuccinate synthetase deficiency.

OBJECTIVE: To monitor long-term survival and outcome of patients with neonatal-onset argininosuccinate synthetase deficiency (ASD) who were treated with specific therapeutic protocols designed to activate alternative pathways of waste nitrogen excretion. DESIGN: Patients for this study included 24 infants born before 1990 and rescued from hyperammonemic coma caused by neonatal-onset ASD; they were referred to this center for enrollment in ongoing clinical studies of sodium benzoate, sodium phenylacetate, and sodium phenylbutyrate. Collaborating physicians throughout the United States and Canada provided information on survival, intellectual development, intercurrent hyperammonemic episodes, and anthropometric and biochemical measurements. RESULTS: The cumulative survival rate was 87.5% at 5 years and 72% at 10 years of age. Survivors include 15 patients currently treated with high doses of sodium phenylbutyrate; two patients have withdrawn. Among the treated group, 11 are classified as severely to profoundly mentally retarded. The remaining four patients have IQ measurements in the borderline to mentally retarded range. All patients have had intercurrent hyperammonemic episodes; our data indicate that the frequency of the episodes has decreased with implementation of the current protocol. These patients are growth retarded, but most have height-for-weight z scores within 2 SD of the mean. Laboratory studies of plasma amino acids and of hematopoietic, renal, and hepatic function are within normal limits with the exception of slightly elevated serum aminotransferase values. CONCLUSION: Our results indicate that these drugs are safe and that the current protocol improves survival rates. However, survival is accompanied by mental retardation, growth retardation, risk of hyperammonemic episodes, and the necessity of lifetime adherence to strict medication and dietary management.

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.

Hyperammonemic coma due to parenteral nutrition in a woman with heterozygous ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency is an X-linked disorder of the urea cycle that can cause hyperammonemic encephalopathy in hemizygous males and heterozygous females. Affected females typically limit protein intake in their diet. This case report describes a 36-year-old woman with ulcerative colitis who went into hyperammonemic coma after administration of total parenteral nutrition. A similar episode of coma had occurred 7 years earlier after she delivered a normal boy. Heterozygous ornithine transcarbamylase deficiency was diagnosed based on a positive allopurinol tolerance test result after elevated levels of plasma glutamine and low plasma citrulline were detected. The protein load associated with parenteral alimentation resulted in symptomatic expression of this partial enzyme deficiency in this unique case. Partial ornithine transcarbamylase deficiency must always be considered in adult women and men with hyperammonemia who have normal liver function test results.

Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial.

Butyrate analogues have been shown to increase fetal hemoglobin (HbF) production in vitro and in vivo. Sodium phenylbutyrate (SPB), an oral agent used to treat individuals with urea-cycle disorders, has been shown to increase HbF in nonanemic individuals and in individuals with sickle cell disease. We have treated eleven patients with homozygous beta thalassemia (three transfusion dependent) and one sickle-beta-thalassemia patient with 20 g/d (forty 500-mg tablets) of SPB for 41 to 460 days. All patients showed an increase in the percent of F reticulocytes associated with treatment, but only four patients responded by increasing their Hb levels by greater than 1 g/dL (mean increase, 2.1 g/dL; range, 1.2 to 2.8 g/dL). None of the transfusion-dependent thalassemia subjects responded. Increase in Hb was associated with an increase in red blood cell number (mean increase, 0.62 x 10(12)/L), and mean corpuscular volume (mean increase, 6 fL). Changes in percent HbF, absolute HbF levels, or alpha- to non-alpha-globin ratios as measured by levels of mRNA and globin protein in peripheral blood did not correlate with response to treatment. Response to treatment was not associated with the type of beta-globin mutation, but baseline erythropoietin levels of greater than 120 mU/mL was seen in all responders and only two of eight nonresponders to SPB. Compliance with treatment was greater than 90% as measured by pill counts. Side effects of the drug included weight gain and/or edema caused by increase salt load in 2/12, transient epigastric discomfort in 7/12, and abnormal body odor in 3/12 subjects. Two splenectomized patients who were not on prophylactic antibiotics developed sepsis while on treatment. We conclude that SPB increases Hb in some patients with thalassemia, but the precise mechanism of action is unknown.

Fetal hemoglobin induction by acetate, a product of butyrate catabolism.

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma-chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF-containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two-carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

Unexpected encephalopathy in chronic renal failure: hyperammonemia complicating acute peritonitis.

A woman with mild chronic renal insufficiency was being treated with glucocorticoids for a presumed chronic inflammatory disease. She developed peritonitis arising from a pelvic abscess, which was drained without complications. Unexpectedly, she became obtunded, and eventually, the neurologic dysfunction was linked to hyperammonemia in spite of normal liver function tests. Hyperammonemia was only transiently controlled in spite of protein restriction, repeated hemodialysis, and the use of biochemical means to reduce ammonia. A recurrent pelvic abscess was drained, and hyperammonemia disappeared. A review of ammonia and nitrogen metabolism indicates that bypassing the liver with shunting of ammonia into the systemic circulation should be added to the causes of symptomatic hyperammonemia. Treatment requires the elimination of the bacteria.

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.