Polycystic kidneys and GM2 gangliosidosis-like disease in neonatal springboks (Antidorcas marsupialis).

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008-2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 µm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 µm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies.

[Evaluation of serum cobalamin concentration in cats with clinical signs of gastrointestinal disease]. / Untersuchungen zur Serumcobalaminkonzentration bei Katzen mit gastrointestinaler Symptomatik.

Medical records of 261 cats presenting with gastrointestinal disease that had a serum cobalamin concentration measured were reviewed. In addition, a reference range for cobalamin (305 - 1.967ng/L) was established using 22 healthy adult cats with undetectable levels of urinary methylmalonic acid. A total of 108 of 261 cats (41.4 %) had hypocobalaminemia; 69 cats (26.4 %) had cobalamin concentrations below the detection limit of the assay (< 150ng/L, group A) and 39 (15 %) had concentrations between 150 - 304ng/L (group B). The remaining 153 (58.6 %) cats had normal cobalamin concentrations (group C). Diarrhea was the most common clinical sign in hypocobalaminemic cats and vomiting or anorexia was the most common sign in normocobalaminemic cats. Only cats with both, vomiting and diarrhea were more likely to have hypocobalaminemia than cats with other clinical signs (odds ratio, 2.879; 95 % CI, 1.313 - 6.310). Serum cobalamin concentration was negatively correlated with age of the patient and positively correlated with body condition score. Cats of group A had a significantly higher neutrophil count (p = 0.0009) and higher MCV (p = 0.0064) and significantly lower hematocrit (p = 0.0018) and albumin concentration (p = 0.0037) than cats in other groups. There was no difference between cats of groups B and C with respect to complete blood cell counts and metabolic profiles. Among the diagnoses made in 125 cats (A 69.6 %, B 59 %, C 35.3 %), lymphoma and inflammatory enteropathy were most common. Lymphoma was diagnosed in 31.2 % (A 53.8 %, B 15.4 %, C 30.8 %) and inflammatory enteropathy in 22.4 % (A 35.7 %, B 7.1 %, C 57.2 %) of cats. Hypocobalaminemia is a frequent problem in cats with gastrointestinal disease. Presenting clinical signs as well as laboratory results may already indicate its probability and severity. However, only values below the detection limit of the assay seem to affect routine bloodwork results. Cobalamin should be routinely measured in feline gastrointestinal disease, as its serum concentration may influence the choice of further diagnostics.

Transient renal Fanconi syndrome in a Chihuahua exposed to Chinese chicken jerky treats.

Transient Fanconi syndrome without azotemia was diagnosed in a dog and was associated with ingestion of Chinese chicken jerky treats. Fanconi syndrome is a proximal renal tubular defect and a diagnosis was made based upon severe glucosuria with normoglycemia, and severe generalized aminoaciduria. The clinical signs of polyuria and polydipsia as well as the massive urinary metabolic abnormalities resolved after jerky treat withdrawal. While frequently seen in North America and Australia, this is the first report of jerky treat induced Fanconi syndrome in continental Europe. Clinicians should be aware of this potential intoxication and be vigilant for a history of jerky treat consumption in a dog with glucosuria.

The spectrum of free neuraminic acid storage disease in childhood: clinical, morphological and biochemical observations in three non-Finnish patients.

N-acetylneuraminic acid (sialic acid) storage disease is a rare autosomal recessive lysosomal disorder. Clinically two major forms exist, an infantile type with severe progression leading to early death, and a milder form (Salla disease) with a protracted course. Intermediate forms may also exist. Diagnosis rests on the determination of an excessive excretion of sialic acid in urine and concomitant storage in fibroblasts, the severe forms exhibiting the highest excretion and storage. We present clinical, morphological, and biochemical data on three non-Finnish patients with sialic acid storage disease. Patient 1 was a preterm infant with neonatal ascites, coarse face, hepatosplenomegaly, pale skin, and wispy hair. Vacuolated lymphocytes were abundant in a peripheral blood smear and he excreted large amounts of free sialic acid. High levels of free sialic acid were also found in cultured skin fibroblasts. He died at age 6 months from progressive respiratory insufficiency. Patient 2 was an 11-month-old Egyptian girl with coarse face, frequent upper respiratory tract infections, hepatosplenomegaly, and severe psycho-motor retardation. Sialic acid excretion was elevated, likewise the storage in fibroblasts. Histological investigations documented vacuolar storage in a skin biopsy and in iliac crest tissue. Patient 3 was a 16-year-old girl with slightly coarse face, severe generalized muscular hypotonia, ataxia, and kyphoscoliosis originally diagnosed as having post-partum asphyxia. She suffered progressive motor function loss and had dysarthria. Urinary sialic acid was elevated and a skin biopsy demonstrated vacuolization. The clinical variability of sialic acid storage disease is exemplified by these three cases. Simple urinary screening for free sialic acid facilitates the diagnosis. The degree of urinary excretion may indeed correlate with clinical presentation and progression.

An improved thin-layer chromatographic method for urinary oligosaccharide screening.

A modified thin-layer chromatographic screening method is presented for the rapid diagnosis of glycoprotein storage disorders based upon excess oligosaccharide excretion. The system has been used successfully in the diagnosis of mannosidosis, GM1 gangliosidosis, mucolipidoses types I and III, aspartyl-glycosaminuria and fucosidosis.

The chemistry of human neonatal femoral epiphyseal cartilage.

The estimation of cartilage constituents such as hydroxyproline and hexosamine may be used to detect gross changes in the total amount of collagen and matrix. This simple approach, which can only be regarded as a "screening" procedure prior to more exhaustive analysis, has been applied to samples of neonatal human cartilage. It is suggested that this method would be useful in analysis cartilage biopsies, especially from patients with bone dysplasias.

Measurement of short-chain acyl-CoA dehydrogenase (SCAD) in cultured skin fibroblasts with hexanoyl-CoA as a competitive inhibitor to eliminate the contribution of medium-chain acyl-CoA dehydrogenase.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency has so far been reported in only very few patients. This is due, in part, to the problems involved in measuring the activity of SCAD unequivocally. The main reason for this difficulty is that butyryl-CoA, the substrate preferably used for SCAD activity measurements, is also dehydrogenated by medium-chain acyl-CoA dehydrogenase (MCAD). Elimination of this contribution can be achieved by means of immune precipitation with a specific MCAD antibody. We now describe a relatively straightforward assay based on the use of gas chromatography/mass spectrometry for detection. The contribution of MCAD to overall butyryl-CoA dehydrogenation was eliminated by adding excess hexanoyl-CoA to the assay medium. The validity of the method developed was checked by SCAD-activity measurements in fibroblasts from an established SCAD-deficient patient.

Stereodifferentiation of 3-hydroxyisobutyric- and 3-aminoisobutyric acid in human urine by enantioselective multidimensional capillary gas chromatography-mass spectrometry.

The chiral metabolites 3-hydroxyisobutyric acid (HIBA) and 3-aminoisobutyric acid (AIBA) are intermediates in the pathways of L-valine and thymine and play an important role in the diagnosis of the very rare inherited metabolic diseases 3-hydroxyisobutyric aciduria (McKusick 236975) and methylmalonic semialdehyde dehydrogenase deficiency (McKusick 603178-MSDD). Until now only a few approaches have been made in enantioselective analysis of HIBA and AIBA and for that reason very little information is available on enantiomeric ratios of these metabolites in man. This paper reports on the simultaneous stereodifferentiation of HIBA and AIBA in human urine as corresponding N(O)-methoxycarbonyl methyl esters by derivatization with methyl chloroformate (MCF) using enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC/MS) with heptakis-(2, 3-di-O-methyl-6-O-tert.-butyl-dimethylsilyl)-beta-cyclodextrin as the chiral stationary phase. During this investigation urine samples from different patients and healthy controls were analyzed in order to reveal characteristic enantiomeric patterns of these metabolites. A trend of dominating R-HIBA excretion in the control urine samples investigated was observed. An excretion of more than 80% S-HIBA was found in the urines of two patients with ketonemic vomiting. There are some clues indicating a possible renal reabsorbtion of S-HIBA similar to those of S-AIBA. Furthermore, there was a significant finding with regard to the enantiomeric distribution of AIBA in a patient with MSDD - a markedly increased excretion of the S-enantiomer in contrast to the other samples. Using the enantiomeric ratios of AIBA, a previously investigated case of benign methylmalonic aciduria (bMMA) could be excluded from the diagnosis of MSDD.

[Skeletal changes in 2 German children with aspartylglycosaminuria]. / Skelettveränderungen bei zwei deutschen Kindern mit Aspartylglucosaminurie.

Two sibs showed delayed speech development, motor retardation and coarsening of their features during their second year of life. Radiological examination of the skeleton showed changes of dysostosis multiplex. Both children showed storage vacuoles in peripheral lymphocytes and a typical oligosaccharide-banded pattern on urinary chromatography. The diagnosis of aspartylglycosaminuria was confirmed by the demonstration of reduced activity of the enzyme aspartylglucosaminidase in leukocytes and in cultured fibroblasts. Treatment of this autosomal recessive inherited glycoprotein storage disease is not possible. Early recognition is important for genetic counselling of the family. This paper describes the first recognised cases in German literature.

SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system.

BACKGROUND: Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds. HYPOTHESIS/OBJECTIVES: To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds. ANIMALS: Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs. METHODS: Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA. RESULTS: In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown. CONCLUSIONS AND CLINICAL IMPORTANCE: These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds.

Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

Ethylmalonic encephalopathy: clinical and biochemical observations.

Ethylmalonic encephalopathy (EE) is a rare, recently defined inborn error of metabolism which affects the brain, gastrointestinal system and peripheral blood vessels and is characterized by a unique constellation of clinical and biochemical features. A 7-month-old male, who presented with psychomotor retardation, chronic diarrhea and relapsing petechiae is described with the objective of highlighting the biochemical and neuroradiological features of this disorder as well as the effect of high-dose riboflavin therapy. Urinary organic acid analysis revealed markedly increased excretion of ethylmalonic acid, isobutyrylglycine, 2-methylbutyrylglycine and isovalerylglycine. Acylcarnitine analysis in dried blood spots showed increased butyrylcarnitine. Short-chain acyl-CoA dehydrogenase (SCAD) activity in muscle was normal as were mitochondrial OXPHOS enzyme activities in cultured skin fibroblasts. In skeletal muscle the catalytic activity of complex II was decreased. Brain MRI revealed bilateral and symmetrical atrophy in the fronto-temporal areas, massive enlargement of the subarachnoid spaces and hyperdensities on T (2) sequences of the basal ganglia. Mutation analysis of the ETHE1 gene demonstrated homozygosity for the Arg163Gly mutation, confirming the diagnosis of EE at a molecular level. On repeat MRI, a significant deterioration was seen, correlating well with the clinical deterioration of the patient.

Simple laboratory determination of excess oligosacchariduria.

I describe a simple set of procedures for the screening of patients' urine to detect oligosaccharide-storage diseases. Urines from patients with mucolipidosis I, mannosidosis, fucosidosis, aspartylglycosaminuria, and type VI glycogen-storage disease can be distinguished by thin-layer chromatography. Patients with beta-galactosidase deficiency can be detected by use of a combination of ion-exchange and thin-layer chromatography. Excess sialyloligosaccharide excretion is detected by using gel filtration and a quantitative assay for neuraminic acid. The advantages of the system are detection of virtually all known disorders in which oligosaccharides are over-excreted, production of characteristic patterns, and small sample requirement.

Urinary oligosaccharide excretion in disorders of glycolipid, glycoprotein and glycogen metabolism. A review of screening for differential diagnosis.

In recent years great interest has centered around metabolic disorders in which excessive oligosacchariduria is a prominent feature. This review describes the methods of both structural and diagnostic investigations of oligosaccharides in a number of these diseases. Special emphasis has been laid upon simple screening methods which would avail themselves to the clinical chemistry laboratory

The simple detection of neuraminic acid-containing urinary oligosaccharides in patients with glycoprotein storage diseases.

Urine samples from patients with different types of glycoprotein storage disease were chromatographed by gel filtration and the fractions analysed for sialic acid. Patients with mucolipidoses I and II excreted the largest amounts of bound sialic acid. One patient with GM1 gangliosidosis showed an abnormal level of sialyloligosaccharide excretion. Other patients showed normal results. With the present method mucolipidoses I and II, together with GM1 gangliosidosis, are readily distinguished from other possible oligosaccharidurias.

Urinary screening for disorders of heteroglycan metabolism. Results of 10 years experience with a comprehensive system.

The results of 10 years experience in urinary screening for disorders of heteroglycan metabolism are presented. Over 5,000 urines were analysed of which 216 were positive for excess mucopolysacchariduria. The enzymatic diagnosis was achieved in 159 mucopolysaccharidoses of which Type III Sanfilippo was the commonest (86 cases), followed by Type II Hunter (31 cases) and Type I Hurler (21 cases). A total of 27 urines were positive for excess oligosacchariduria, the enzymatic diagnosis being established in 20 cases. The most frequently encountered oligosaccharidosis was GM1 gangliosidosis (10 cases), followed by mannosidosis (5 cases). No cases of fucosidosis were found.

Acylcarnitines in intermediary metabolism.

From the time of its discovery in 1905 until the first description of its deficiency in 1973, the role of carnitine in intermediary metabolism was decidedly vague. Identification of carnitine acyl transferases and their products, acylcarnitines, have paved the way to the confirmation of the importance of carnitine in the transfer of fatty acid CoAs into the mitochondrion for beta-oxidation and energy production. The elucidation of defects in fatty acid oxidation together with the concept of carnitine therapy in certain organoacidaemias have given a new meaning to the term acylcarnitine. Not only are these compounds of diagnostic importance, their formation may be part of a secondary carnitine depletion which may be brought about as a result of various medications. Recent evidence suggests that long-chain acylcarnitines are responsible for cardiac arrhythmias and other effects, both good and bad, will certainly be found. This review will attempt to highlight the importance of acylcarnitines, from their production, the difficulties in analysis, the diagnostic possibilities and their positive and negative effects on intermediary metabolism.