HOMOCARNOSINOSIS: INCREASED CONTENT OF HOMOCARNOSINE AND DEFICIENCY OF HOMOCARNOSINASE IN BRAIN

Biochemical studies were carried out on a brain biopsy obtained from a 40 y old woman with homocarnosinosis. This patient and 2 of her siblings have a progressive neurological disorder characterized by spastic paraplegia, dementia and retinal pigmentation. Homocarnosine concentrations in their CSF, as well as in the CSF of their neurologically normal mother, are elevated 20-40-fold. Homocarnosine content was 4 times higher in the patient's frontal cortex than in biopsied cortex from a large group of control subjects. The activity of the synthetic enzyme, homocarnosine-carnosine synthetase. was not increased in the homocarnosinosis patient's brain. Homocarnosinase activity, however, was undetectable in the patient's brain biopsy, while it could readily be measured in comparable biopsies from control subjects. We conclude that homocarnosinase deficiency is probably inhcrited as an autosomal recessive trait, but that it is unlikely of itself to have caused the neurological syndrome affecting our patients.     

Homocarnosinosis: Hypercarnosinuria

Abstract: Homocarnosine (γ-aminobutyrylhistidine) is a brain-specific dipeptide. Homocarnosinosis is a familial metabolic disorder in which spastic paraplegia, progressive mental deficiency, and retinal pigmentation coexist with increased CSF homocarnosine levels, i.e. approximately 20 times higher than the mean control level. In the present study, the urinary excretion of carnosine ( β -alanylhistidine) and anserine ( β -alanyl-1-methylhistidine) was determined in patients with homocarnosinosis and in their close relatives. Both the patients and their relatives were also loaded with chicken meat, which is rich in anserine and carnosine. The results were compared with those obtained in childhood hypercarnosinuria with serum carnosinase deficiency. Somewhat surprisingly, patients with homocarnosinosis were also shown to have hypercarnosinuria. Chicken meat loading in healthy individuals results in increased excretion of carnosine and anserine and, in addition, 1-methylhistidine in the urine. Homocarnosinosis patients and patients with serum carnosinase deficiency also showed an increased excretion of carnosine and anserine, but 1-methylhistidine was not detected in serum carnosinase deficiency, and it was present only in very small amounts in homocarnosinosis. This defect seems to be due to lack of the hydrolyzing enzyme activity. The biochemical and clinical similarities between adult homocarnosinosis and infantile serum carnosinase deficiency are intriguing. A complete insight into the relationship between them must await further investigation.     

Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease

The importance of intracellular folate metabolism is illustrated by the severity of symptoms and complications caused by inborn disorders of folate metabolism or by folate deficiency. We examined three children of healthy, distantly related parents presenting with megaloblastic anemia and cerebral folate deficiency causing neurologic disease with atypical childhood absence epilepsy. Genome-wide homozygosity mapping revealed a candidate region on chromosome 5 including the dihydrofolate reductase (DHFR) locus. DHFR sequencing revealed a homozygous DHFR mutation, c.458A>T (p.Asp153Val), in all siblings. The patients' folate profile in red blood cells (RBC), plasma, and cerebrospinal fluid (CSF), analyzed by liquid chromatography tandem mass spectrometry, was compatible with DHFR deficiency. DHFR activity and fluorescein-labeled methotrexate (FMTX) binding were severely reduced in EBV-immortalized lymphoblastoid cells of all patients. Heterozygous cells displayed intermediate DHFR activity and FMTX binding. RT-PCR of DHFR mRNA revealed no differences between wild-type and DHFR mutation-carrying cells, whereas protein expression was reduced in cells with the DHFR mutation. Treatment with folinic acid resulted in the resolution of hematological abnormalities, normalization of CSF folate levels, and improvement of neurological symptoms. In conclusion, the homozygous DHFR mutation p.Asp153Val causes DHFR deficiency and leads to a complex hematological and neurological disease that can be successfully treated with folinic acid. DHFR is necessary for maintaining sufficient CSF and RBC folate levels, even in the presence of adequate nutritional folate supply and normal plasma folate.     

A novel, quantitative assay for homocarnosine in cerebrospinal fluid using stable-isotope dilution liquid chromatography-tandem mass spectrometry

We describe a rapid and sensitive method for the quantification of homocarnosine in physiological fluids, with particular emphasis on cerebrospinal fluid (CSF). Homocarnosine was quantified as the butyl derivative, with (2)H(2)-l-homocarnosine as internal standard. Following deproteinization of CSF samples, supernatants were evaporated to dryness and derivatized with 10% 6M HCl in butanol. Samples were chromatographed on a C(18) column and detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in the multiple reaction monitoring mode. The intra- and inter-assay variations were 4.6 and 10.9%, respectively. Mean recovery of homocarnosine at two concentrations was 105%. The limit of detection in CSF approximated 20 nmol/L. CSF homocarnosine is age dependent and ranges from <0.02 to 10 micromol/L. Our method is applicable to the analysis of CSF derived from patients with heritable defects in the GABA pathway, patients with homocarnosinosis or serum carnosinase deficiency, and should be applicable to other model systems in order to further explore the biological role and significance of homocarnosine in mammalian systems.     

Purine nucleoside phosphorylase deficiency: a mutation update

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.     

14-3-3 Protein, Total Tau and Phosphorylated Tau in Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease and Neurodegenerative Disease in Japan

1.Sporadic Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal disease. Patients with CJD usually become akinetic mutism within approximately 6 months. In addition, clinical signs and symptoms at early stage of sporadic CJD may not be easy to distinguish from other neurodegenerative diseases by neurological findings. However, diagnostic biochemical parameters including 14-3-3 protein, S100, neuron-specific enorase in cerebrospinal fluid (CSF) have been used as diagnostic markers, elevated titers of these markers can also be observed in CSF in other neurodegenerative diseases. Therefore, we examined other biochemical markers to discriminate CJD from other neurodegenerative diseases in CSF. 2.We analyzed CSF samples derived from 100 patients with various neurodegenerative disorders by Western blot of 14-3-3 protein, quantification of total tau (t-tau) protein, and phosphorylated tau (p-tau) protein. All patients with CJD in this study showed positive 14-3-3 protein and elevated t-tau protein (>1000 pg/mL) in CSF. We also detected positive 14-3-3 protein bands in two patients in non-CJD group (patients with dementia of Alzheimer's type; DAT) and also detected elevated t-tau protein in three patients in non-CJD group. Elevated t-tau protein levels were observed in two patients with DAT and in one patient with cerevrovascular disease in acute phase. 3.To distinguish patients with CJD from non-CJD patients with elevated t-tau protein in CSF, we compared the ratio of p-tau and t-tau proteins. The p-/t-tau ratio was dramatically and significantly higher in DAT patients rather than in CJD patients. 4.Therefore, we concluded that the assay of t-tau protein may be useful as 1st screening and the ratio of p-tau protein/t-tau protein would be useful as 2nd screening to discriminate CJD from other neurodegenerative diseases.     

Minocycline effects on the cerebrospinal fluid proteome of experimental autoimmune encephalomyelitis rats

To identify response biomarkers for pharmaceutical treatment of multiple sclerosis, we induced experimental autoimmune encephalomyelitis (EAE) in rats and treated symptomatic animals with minocycline. Cerebrospinal fluid (CSF) samples were collected 14 days after EAE induction at the peak of neurological symptoms, and proteomics analysis was performed using nano-LC-Orbitrap mass spectrometry. Additionally, the minocycline concentration in CSF was determined using quantitative matrix-assisted laser desorption/ionization-triple-quadrupole tandem mass spectrometry (MALDI-MS/MS) in the selected reaction monitoring (SRM) mode. Fifty percent of the minocycline-treated EAE animals did not show neurological symptoms on day 14 ("responders"), while the other half displayed neurological symptoms ("nonresponders"), indicating that minocycline delayed disease onset and attenuated disease severity in some, but not all, animals. Neither CSF nor plasma minocycline concentrations correlated with the onset of symptoms or disease severity. Analysis of the proteomics data resulted in a list of 20 differentially abundant proteins between the untreated animals and the responder group of animals. Two of these proteins, complement C3 and carboxypeptidase B2, were validated by quantitative LC-MS/MS in the SRM mode. Differences in the CSF proteome between untreated EAE animals and minocycline-treated responders were similar to the differences between minocycline-treated responders and nonresponders (70% overlap). Six proteins that remained unchanged in the minocycline-treated animals but were elevated in untreated EAE animals may be related to the mechanism of action of minocycline.     

Purine Nucleoside Phosphorylase Deficiency: A Mutation Update

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.     

Identification of a Biomarker in Cerebrospinal Fluid for Neuronopathic Forms of Gaucher Disease

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available.     

Adrenal and testicular function in 14 patients with adrenoleukodystrophy or adrenomyeloneuropathy

Testicular and adrenal function were evaluated in 12 patients with adrenoleukodystrophy and 2 patients with adrenomyeloneuropathy. Although only 5 subjects had clinical symptoms suggesting adrenal insufficiency, an additional 5 showed laboratory evidence of reduced adrenal reserve. 9 of the 14 patients developed neurological deficits prior to the onset of clinical or biological adrenal insufficiency. In the remaining 5 patients, adrenal insufficiency antedated the appearance of neurological symptoms; 2 of these 5 patients had only laboratory evidence of hypoadrenocorticism, and 3 had both clinical and laboratory abnormalities. None of the prepubertal patients had detectable signs of testicular insufficiency, while 3 of the 7 pubertal/adult patients had elevated serum LH or FSH levels. This mild testicular deficiency was seen only in association with clinical adrenal insufficiency and significant neurological impairment.     

Nucleotide Degradation Products in Cerebrospinal Fluid (CSF) in Inherited and Acquired Pathologies

CSF purines were grossly elevated compared with controls only in adenylosuccinate lyase (ADSL) deficiency and TB meningitis. The former representing low permeability, the latter severe damage to the normal blood/brain barrier. By contrast, the similarity to controls, with no difference between Lesch–Nyhan disease (LND) or LND variants, would exclude hypoxia as a factor in the severe neurological deficits in LND. Similar findings in purine nucleoside phosphorylase (PNP) deficiency (although nucleosides replace the normal bases) likewise exclude hypoxia in the aetiology of the albeit milder neurological deficits.     

Nucleotide degradation products in cerebrospinal fluid (CSF) in inherited and acquired pathologies

CSF purines were grossly elevated compared with controls only in adenylosuccinate lyase (ADSL) deficiency and TB meningitis. The former representing low permeability, the latter severe damage to the normal blood/brain barrier. By contrast, the similarity to controls, with no difference between Lesch-Nyhan disease (LND) or LND variants, would exclude hypoxia as a factor in the severe neurological deficits in LND. Similar findings in purine nucleoside phosphorylase (PNP) deficiency (although nucleosides replace the normal bases) likewise exclude hypoxia in the aetiology of the albeit milder neurological deficits.     

Assay and biochemical characterization of angiotensin-I-converting enzyme in cerebrospinal fluid

A spectrofluorimetric method was adapted for determination of angiotensin-I-converting enzyme (ACE) in untreated native cerebrospinal fluid (CSF). Benzyloxycarbonyl-phenylalanyl-histidyl-leucine was applied as enzyme substrate. The biochemical behavior of ACE of CSF (CACE) was studied. The pH optimum was found to be 8.0 using borax phosphate buffer. The determination of Km was 10.7 +/- 3.3 (SD) mumol/l. ACE could be blocked by 8-hydroxyquinoline (100%) and phenanthroline (80%), but only slight inhibition was observed by teprotide (43%), EDTA (40%) and captopril (31%). The influence of various drugs on CACE was also tested. The different biochemical behavior of CACE compared to serum ACE suggested that an isoenzyme exists in CSF. CACE has been found to be elevated in neuroimmunological and inflammatory disorders of the nervous system compared to levels in healthy controls.     

Clinical symptoms and biochemical properties of three new glucosephosphate isomerase variants

Glucosephosphate isomerase deficiency as the cause of macrocytic congenital nonspherocytic hemolytic anemia is described in three unrelated families. The biochemical properties of the variant glucosephosphate isomerases indicate that the patients have new variants, designated as GPI Kiel, GPI Hamburg, and GPI Homburg. The severity of the clinical symptoms depended on the amount of residual GPI activity and the biochemical properties of the variant enzyme. Thus the patient with GPI Kiel (34% residual activity) whose variant GPI was slightly unstable showed a mild chronic hemolytic anemia. The patient with GPI Homburg (7% residual activity) whose variant enzyme was stable and had a reduced specific activity, suffered from severe congenital hemolytic anemia and neuromuscular symptoms. Due to the special properties of GPI Homburg, we assume that both the hematological and neuromuscular symptoms of the patient with GPI Homburg are caused by his GPI deficiency. The twins with GPI Hamburg (27% residual activity) had a distinctly unstable variant enzyme and had suffered from hemolytic crises since birth. Only GPI Homburg showed an altered electrophoretic mobility and an increased affinity for fructose-6-phosphate. The other two variants had normal values.     

Free-GABA levels in the cerebrospinal fluid of patients suffering from several neurological diseases Its potential use for the diagnosis of diseases which course with inflammation and tissular necrosis

Summary Free GABA levels were measured in the cerebrospinal fluid (CSF) of 74 neurological patients suffering from cerebral cysticercosis (n = 9), Parkinson's disease (n = 5), multiple sclerosis (n = 6), epilepsy (n = 24), meningeal tuberculosis (n = 6), viral encephalitis (n = 3), cerebrovascular disease (n = 8) and several kinds of dystonia (n = 5). A statistical significant four-fold elevation in free GABA levels was found in patients with cerebral cysticercosis. A non statistical significant two-fold increase in free GABA levels was also encountered in the CSF of patients affected by cerebrovascular disease and viral encephalitis. No changes in CSF free GABA levels were found in patients suffering from any of the other disorders. It is suggested that free GABA levels may be elevated in the CSF of patients suffering from neurological diseases which course with inflammation and tissular necrosis such as cerebral cysticercosis. Much work is needed however to establishd whether CSF free GABA levels can be used as a diagnostic tool in at least some type of these patients.     

Relationship between photosynthetic pigments and chlorophyll fluorescence in soybean under varying phosphorus nutrition at ambient and elevated CO<Subscript>2</Subscript>

To assess the relationship between chlorophyll (Chl) fluorescence (CF) and photosynthetic pigments, soybean was grown under varying phosphorus (P) nutrition at ambient and elevated CO₂ (EC). The EC stimulated, but P deficiency decreased plant height, node numbers, and leaf area concomitantly with the rates of stem elongation, node addition, and leaf area expansion. Under P deficiency, CF parameters and pigments declined except that carotenoids (Car) were relatively stable indicating its role in photoprotection. The CF parameters were strongly related with Chl concentration but not with Chl a/b or Car. However, total Chl/Car showed the strongest association with CF parameters such as quantum efficiency and yield of photosystem II. This relationship was not affected by CO₂ treatment. The high correlation between CF and total Chl/Car underscores the significance of the quantification of both, Chl and Car concentrations, to understand the photochemistry and underlying processes of photoprotection and mechanisms of excess energy dissipation in a given environment.     

Critical evaluation of the biological role of IgM in cerebrospinal fluid in inflammatory and other diseases of the nervous system

In a group of 10,156 patients with neurological diseases, the IgM level was assessed (using laser nephelometry) both in cerebrospinal fluid (CSF) and serum; concentration of other 17 protein fractions was also simultaneously determined: albumin, immunoglobulins, acute phase reactants, complement components, apolipoproteins, proteinase inhibitors and alpha1-microglobulin. Total protein, element counts and glucose level were also evaluated. In patients with normal CSF findings, only limited statistically significant correlations were demonstrated between IgM and other CSF protein fractions while, in the group of patients with pathological CSF findings, significant correlations were found between CSF(IgM) and other immunoglobulins, complement fractions and the rate of intrathecal synthesis of immunoglobulins. Correlations were also found between CSF(IgM) and CSF antithrombin-III and alpha1-microglobulin. Correlations between CSF(IgM) and CSF apolipoproteins support the theory of CNS tissue destruction whenever the concentration of CSF apolipoproteins is elevated. Our data substantially contribute to establishing the diagnosis in patients with neurological diseases; simultaneous measurement of a high number of CSF proteins is becoming inevitable for a reasonable assessment of the CSF Protein Status.     

Chronic meningitis and Lyme disease in Sweden.

We studied 35 patients with chronic meningitis. The neurological abnormalities included aseptic meningitis, cranial neuropathy (mostly facial palsy), motor and sensory peripheral radiculoneuropathy, and myelitis. Neurological symptoms were sometimes preceded by erythema chronicum migrans or an insect bite and were often accompanied by fever, malaise, profound fatigue, and weight loss. The cerebrospinal fluid (CSF) abnormalities consisted of a predominantly mononuclear pleocytosis, an elevated CSF protein (mean 2.3 g/l), intrathecal synthesis of oligoclonal immunoglobulin G, and, in half of the patients, a fall in the CSF/blood glucose ratio. High antibody titers to the Lyme spirochete and the Swedish Ixodes ricinus spirochete were demonstrated by immunofluorescence in 26 of the 35 patients. By imprint immunofixation of electrofocused samples of serum and CSF, intrathecal production of oligoclonal Lyme-spirochete-specific IgG was demonstrated in one patient with chronic meningitis. Four sequential paired samples of serum and CSF from this patient showed local synthesis of spirochete-specific antibodies in CSF. The 35 patients improved or recovered, sometimes dramatically, during a two-week course of intravenous penicillin G.     

Heterogeneity of carotenoid content and composition in LH2 of the purple sulphur bacterium Allochromatium minutissimum grown under carotenoid-biosynthesis inhibition

The effects brought about by growing Allochromatium (Alc.) minutissimum in the presence of different concentrations of the carotenoid (Car) biosynthetic inhibitor diphenylamine (DPA) have been investigated. A decrease of Car content (from approximately 70% to >5%) in the membranes was accompanied by an increase of the percentage of (immature) Cars with reduced numbers of conjugated C=C bonds (from neurosporene to phytoene). Based on the obtained results and the analysis of literature data, the conclusion is reached that accumulation of phytoene during inhibition did not occur. Surprisingly, DPA inhibited phytoene synthase instead of phytoene desaturase as generally assumed. The distribution of Cars in peripheral antenna (LH2) complexes and their effect on the stability of LH2 has been investigated using absorption spectroscopy and HPLC analysis. Heterogeneity of Car composition and contents in the LH2 pool is revealed. The Car contents in LH2 varied widely from control levels to complete absence. According to common view, the assembly of LH2 occurs only in the presence of Cars. Here, we show that the LH2 can be assembled without any Cars. The presence of Cars, however, is important for structural stability of LH2 complexes.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine deficiency encephalopathy: 3. Transketolase

Chronic thiamine deprivation in the rat leads to ataxia, loss of righting reflex and neuropathological damage to lateral vestibular nucleus. Before onset of neurological symptoms, transketolase (TK) activities were found to be selectively reduced by 25% in lateral vestibular nucleus and surrounding pons. Further progression of thiamine deprivation resulted in a generalized reduction in TK activity. Measurement of enzyme activity in the presence of added TPP cofactor in vitro did not lead to normalisation of enzyme activities suggesting loss of apoenzyme. Administration of thiamine to symptomatic thiamine-deprived rats resulted in reversal of neurological symptoms and to normalisation of defective TK activities in less vulnerable structures such as cerebral cortex striatum and hippocampus; reduction of TK activity, however, persisted in brainstem and cerebellar regions. Pyrithiamine treatment results, within 3 weeks, in loss of righting reflex, convulsions and more widespread neuropathological damage compared to that observed following thiamine deprivation. TK activity was found to be significantly decreased before the onset of neurological symptoms in all brain regions and appearance of symptoms was accompanied by more severe reductions of TK. In contrast to chronic thiamine deprivation, TK activities following pyrithiamine treatment were: (i) equally reduced in magnitude in vulnerable and non-vulnerable brain structures, (ii) unchanged following reversal of neurological abnormalities by thiamine administration.