l-Carnitine prevents oxidative stress in striatum of glutaryl-CoA dehydrogenase deficient mice submitted to lysine overload.

The deficiency of the enzyme glutaryl-CoA dehydrogenase leads to predominant accumulation of glutaric acid (GA) in the organism and is known as glutaric acidemia type I (GA1). Despite the mechanisms of brain damage involved in GA1 are not fully understood, oxidative stress may be involved in this process. Treatment is based on protein/lysine (Lys) restriction and l-carnitine (L-car) supplementation. L-car was recently shown to have an important antioxidant role. A knockout mice model (Gcdh-/-) submitted to a dietary overload of Lys was developed to better understand the GA1 pathogenesis. In this study, we evaluated L-car and glutarylcarnitine levels, the lipid and protein damage, reactive oxygen species (ROS) production and antioxidant enzymes activities in striatum of Gcdh-/- and wild-type (WT) mice. We also determined the effect of the L-car treatment on these parameters. Thirty-day-old Gcdh-/- and WT mice were fed a normal chow (0.9% Lys) or submitted to a high Lys diet (4.7%) for 72 h. Additionally, these animals were administered with three intraperitoneal injections of saline or L-car in different times. Gcdh-/- mice were deficient in L-car and presented a higher glutarylcarnitine levels. They also presented lipid and protein damage, an increased ROS production and altered antioxidant enzymes compared to WT mice. Additionally, mice exposed to Lys overload presented higher alterations in these parameters than mice under normal diet, which were significantly decreased or normalized in those receiving L-car. Thus, we demonstrated a new beneficial effect of the L-car treatment attenuating or abolishing the oxidative stress process in Gcdh-/- mice.

Methylmalonic Aciduria Secondary to Selective Cobalamin Malabsorption in a Yorkshire Terrier.

An 8 wk old male Yorkshire terrier was presented with a 2 wk history of recurrent hypoglycemia, lethargy, and seizures. Investigations revealed a marked increase in blood ammonia, low serum cobalamin, and increased levels of urinary methylmalonic acid (MMA) excretion. No liver vascular abnormality was detected. The patient was diagnosed with methylmalonic aciduria due to cobalamin malabsorption. The patient responded well to parenteral cobalamin administration, and the urinary MMA levels normalized rapidly following instigation of treatment. Due to the suspected hereditary nature of selective cobalamin deficiency, one sibling of this dog was screened and found to be normal. This is the first reported case of MMA secondary to hypocobalaminemia in Yorkshire terriers, and the second report of this disease in a dog in the United Kingdom. Given the fact that clinical signs of MMA are similar to those seen in dogs with portosystemic shunts and that Yorkshire terriers are predisposed to liver vascular abnormalities, this case report adds important clinical information to the current available literature.

Serum cobalamin and methylmalonic acid concentrations in dogs with chronic gastrointestinal disease.

OBJECTIVE: To determine the prevalence of hypocobalaminemia or methylmalonic acidemia (or both) in dogs with chronic gastrointestinal disease. SAMPLE: Serum samples from 56 dogs with chronic gastrointestinal disease and 43 control dogs. PROCEDURES: Serum cobalamin and methylmalonic acid (MMA) concentrations were measured in all samples and compared between groups. A correlation between serum cobalamin and MMA concentrations and the canine chronic enteropathy clinical activity index was evaluated via the Spearman rank correlation. RESULTS: 20 of 56 (36%) dogs with gastrointestinal disease had hypocobalaminemia. Serum cobalamin concentrations were significantly lower in dogs with gastrointestinal disease than in control dogs. Five of 56 (9%) dogs with chronic gastrointestinal disease and 5 of 20 (25%) hypocobalaminemic dogs had increased MMA concentrations. There was a significant negative correlation (Spearman r = -0.450) between serum cobalamin and MMA concentrations in dogs with gastrointestinal disease. No correlation was found between the canine chronic enteropathy clinical activity index and serum cobalamin or MMA concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicated the prevalence of hypocobalaminemia in dogs with chronic gastrointestinal disease was 20 of 56 (36%). Five of 20 (25%) hypocobalaminemic dogs had increased serum MMA concentrations, which indicated that although hypocobalaminemia was common in these dogs, it did not always appear to be associated with a deficiency of cobalamin on a cellular level. Hypocobalaminemia is a risk factor for negative outcome in dogs with chronic gastrointestinal disease and should be considered in every patient with corresponding clinical signs.

Serum cobalamin, urine methylmalonic acid, and plasma total homocysteine concentrations in Border Collies and dogs of other breeds.

OBJECTIVE: To determine reference ranges for serum cobalamin (Cbl), urine methylmalonic acid (uMMA), and plasma total homocysteine (tHcys) concentrations and to compare values for healthy control dogs with values for Border Collies (BCs), a breed in which hereditary cobalamin deficiency has been identified. ANIMALS: 113 BCs, 35 healthy control dogs fed a typical diet, and 12 healthy dogs fed a bone and raw food diet exclusively. PROCEDURES: Urine and blood samples were obtained from each dog and Cbl, uMMA, and tHcys concentrations were determined. RESULTS: Reference ranges for Cbl (261 to 1,001 ng/L), uMMA (0 to 4.2 mmol/mol of creatinine), and tHcys (4.3 to 18.4 µmol/L) concentrations were determined. Four BCs had a Cbl concentration lower than the assay detection limit (150 ng/L); median uMMA and tHcys concentrations in these dogs were 4,064 mmol/mol of creatinine and 51.5 µmol/L, respectively. Clinical abnormalities included stunted growth, lethargy, anemia, and proteinuria. Abnormalities improved after administration of cobalamin. Of the 109 healthy BCs with Cbl and tHcys concentrations within reference ranges, 41 (37.6%) had a high uMMA concentration (range, 5 to 360 mmol/mol). Results for dogs fed raw food were similar to those for control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Hereditary cobalamin deficiency is a rare disease with various clinical signs. The finding of methylmalonic aciduria in healthy eucobalaminemic BCs and BCs with clinical signs of Cbl deficiency was surprising and indicated these dogs may have defects in intracellular processing of Cbl or intestinal Cbl malabsorption, respectively. Studies investigating Cbl absorption and metabolic pathways are warranted.

Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen.

The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.

L-2-hydroxyglutaric aciduria: characterisation of the molecular defect in a spontaneous canine model.

l-2-hydroxyglutaric aciduria (l-2-HGA) is a neurometabolic disorder that produces a variety of clinical neurological deficits, including psychomotor retardation, seizures and ataxia. The biochemical hallmark of l-2-HGA is the accumulation of l-2-hydroxyglutaric acid (l-2-HG) in cerebrospinal fluid, plasma and urine. Mutations within the gene L2HGDH (Entrez Gene ID 79944) on chromosome 14q22 encoding L-2-hydroxyglutaric acid dehydrogenase have recently been shown to cause l-2-HGA in humans. Using a candidate gene approach in an outbred pet dog population segregating l-2-HGA, the causal molecular defect was identified in the canine homologue of L2HGDH and characterised. DNA sequencing and pedigree analysis indicate a common founder effect in the canine model. The canine model shares many of the clinical and MRI features of the disease in humans and represents a valuable resource as a spontaneous model of l-2-HGA.

Prenatal diagnosis of bovine citrullinaemia.

Holstein/Friesian embryos, progeny of citrullinaemia heterozygotes, were transferred to recipient cows. The citrullinaemia genotype of the fetus/calf was established, at birth, by estimation of citrulline concentration in plasma or by analyses of argininosuccinate synthetase exon 5 sequences in DNA from blood leucocytes. Between 115 and 125 days of gestation amniotic fluid was collected transabdominally from nine recipient cows. The mean citrulline concentration in amniotic fluid from fetuses unaffected with citrullinaemia was 28 microM, SD 9 and range 15 to 38 microM. Citrulline concentration was 107 and 130 microM in amniotic fluid from two fetuses homozygous for the citrullinaemia mutation. Only the normal bovine citrullinaemia sequence was detected in cells cultured from amniotic fluid taken from two homozygous normal and two heterozygous fetuses. Both normal and mutant sequences were found in cells cultured from amniotic fluid collected from three of five heterozygous fetuses. Only the mutant sequence was found in cells cultured from the two citrullinaemia affected fetuses. MHC class II DRB3 haplotyping revealed that in four instances cultured cells were from the surrogate mother, not the fetus, these included samples from two heterozygotes in which only the normal sequence was detected in amniotic fluid.

Cobalamin deficiency associated with methylmalonic acidemia in a cat.

A 9-month-old sexually intact male longhair cat was examined because of lethargy, anorexia, cold intolerance, and failure to thrive since acquisition at an early age. Clinical signs of disease were less pronounced when the cat was fed a low-protein diet. Anemia, hypoglycemia, low total CO2 content, and hyperammonemia were detected. The cat was euthanatized. Urine obtained immediately before euthanasia contained a large amount of methylmalonic acid. Total serum cobalamin concentration was low. Hepatic methylmalonic-CoA mutase activity, with and without the addition of coenzyme adenosylcobalamin, was consistent with a cobalamin deficiency. Methylmalonic acidemia secondary to a putative defect in cobalamin absorption was diagnosed.

Molecular definition of bovine argininosuccinate synthetase deficiency.

Citrullinemia is an inborn error of metabolism due to deficiency of the urea cycle enzyme, argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5]. The disease was first described in humans but was recently reported in dairy cattle in Australia. Here we report the nucleotide sequence of the normal bovine cDNA for argininosuccinate synthetase and the mutation present in animals with citrullinemia. Analysis of DNA from affected animals by Southern blotting did not readily identify the mutation in the bovine gene. RNA (Northern) blotting revealed a major reduction in the steady-state amount of mRNA in the liver of affected animals to less than 5% of controls. The bovine cDNA was cloned and sequenced and revealed 96% identity with the deduced human sequence at the amino acid level. Starting with mutant bovine liver, the mRNA was reverse-transcribed; the cDNA product was amplified with the polymerase chain reaction, cloned, and sequenced. The sequence revealed a C----T transition converting arginine-86 (CGA) to a nonsense codon (TGA). A second C----T transition represented a polymorphism in proline-175 (CCC----CCT). The mutation and the polymorphism were confirmed by amplification of genomic DNA and demonstration with restriction endonuclease enzymes of both the loss of an Ava II site in DNA from mutant animals at codon 86 and the presence or absence of a Dde I site at codon 175. The loss of the Ava II site can be used for rapid, economical, nonradioactive detection of heterozygotes for bovine citrullinemia.

Ultrastructural findings in citrullinaemia in Holstein-Friesian calves.

Ultrastructural findings in the brains and livers of four calves affected with citrullinaemia due to a hereditary deficiency of the urea cycle enzyme arginosuccinate synthetase are described. The calves were affected in the 1st week of life with severe neurological disease characterised by depression, head pressing, stupor, convulsions and coma, accompanied by marked elevation of plasma citrulline and increasing plasma ammonia levels. Lesions included mild to moderate diffuse astroglial oedema in the cerebrocortical grey matter, and mild to severe hepatocellular hydropic change. The onset of the severe neurological signs was correlated with increasing levels of plasma ammonia, and the cerebral lesions were considered consistent with a bovine hyperammonaemic encephalopathy.

Increase of Na-K-ATPase activity, glutamate, and aspartate uptake in dog erythrocytes associated with hereditary high accumulation of GSH, glutamate, glutamine, and aspartate.

We have found convincing evidence for the presence of Na-K-ATPase and high potassium (K) and low sodium (Na) concentrations in the erythrocytes of some dogs associated with hereditary high concentrations of erythrocyte glutathione and some amino acids, glutamate, glutamine, and aspartate. The Na-K-ATPase activity of the erythrocyte membranes of the dogs was about 3 times that of human erythrocyte membranes, whereas the enzyme activity was not detected in control dogs with a normal level of blood glutathione. The Michaelis constant of the enzyme for ATP (Km ATP) was 6.6 X 10(-3)M in the dogs' erythrocytes and 5.0 X 10(-4)M in the human erythrocytes. The concentration of K in the erythrocytes in the dogs examined was about 11 times that of the controls, whereas the erythrocyte Na concentration in the dogs was about one-third that of the controls. The concentrations of K and Na in the plasma of the dogs were equal to those of the controls. Furthermore, L-3H-glutamate and L-3H-aspartate uptake by those cells with high activity of Na-K-ATPase greatly increased, while L-3H-glutamine uptake was unchanged. It appeared that Na+ and K+ gradients created by Na-K-ATPase across the cell membrane might stimulate glutamate and aspartate uptake by the cells, thus causing the high accumulation of such amino acids in the cells.

Tyrosine aminotransferase deficiency in mink (Mustela vision): a model for human tyrosinemia II.

Mink pseudodistemper, a recessive disease associated with high blood tyrosine levels, is an animal analogue of the human inborn error of metabolism, tyrosinemia II. Affected mink and man have eye and skin lesions. Affected mink have no hepatic tyrosine aminotransferase (TAT) activity, as measured immunologically and biochemically. Hepatic mitochondrial aspartate aminotransferase is increased to 188% of control. This new genetic animal model of TAT deficiency should allow new studies of tyrosine metabolism.

Inherited metabolic disease in laboratory animals: a review.

Research on the screening for and study of animal models of inherited metabolic disease is reviewed. It is emphasized that an animal model, to be of value, must be an inherited deficiency of the same enzyme as the one deficient in the human syndrome. If this criterion is adhered to there is a remarkable identity in aetiology between animal and man. Specific examples of inherited metabolic disease in laboratory animals are described for: amino acid metabolism; lysosomal storage diseases, carbohydrate metabolism, transport disorders and trace element metabolism; the mutants found in mice being the easiest to manipulate biochemically and genetically. There is still a lack of adequate screening programmes for animal homologues of the more serious human inborn errors (such as lysosomal storage diseases) where laboratory studies could provide significant advances in therapy.

A syndrome of hereditary tyrosinemia in mink (Mustela vison Schreb.).

A hereditary disease in mink (Mustela vison Schreb.) leading to death when the affected kits are about six weeks old has been investigated. The disorder is inherited as a simple autosomal recessive character. Strongly elevated plasma tyrosine concentration is an outstanding feature of the disease. An enzyme defect in tyrosine aminotransferase (EC 2.6.1.5) or 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) is considered together with the possibility of a parallel between the disease in mink and the disease tyrosinosis or hereditary tyrosinemia in man.