A 9-yr evaluation of carrier erythrocyte encapsulated adenosine deaminase (ADA) therapy in a patient with adult-type ADA deficiency.

Adenosine deaminase (ADA) deficiency is an inherited disorder which leads to elevated cellular levels of deoxyadenosine triphosphate (dATP) and systemic accumulation of its precursor, 2-deoxyadenosine. These metabolites impair lymphocyte function, and inactivate S-adenosylhomocysteine hydrolase (SAHH) respectively, leading to severe immunodeficiency. Enzyme replacement therapy with polyethylene glycol-conjugated ADA is available, but its efficacy is reduced by anti-ADA neutralising antibody formation. We report here carrier erythrocyte encapsulated native ADA therapy in an adult-type ADA deficient patient. Encapsulated enzyme is protected from antigenic responses and therapeutic activities are sustained. ADA-loaded autologous carrier erythrocytes were prepared using a hypo-osmotic dialysis procedure. Over a 9-yr period 225 treatment cycles were administered at 2-3 weekly intervals. Therapeutic efficacy was determined by monitoring immunological and metabolic parameters. After 9 yr of therapy, erythrocyte dATP concentration ranged between 24 and 44 micromol/L (diagnosis, 234) and SAHH activity between 1.69 and 2.29 nmol/h/mg haemoglobin (diagnosis, 0.34). Erythrocyte ADA activities were above the reference range of 40-100 nmol/h/mg haemoglobin (0 at diagnosis). Initial increases in absolute lymphocyte counts were not sustained; however, despite subnormal circulating CD20(+) cell numbers, serum immunoglobulin levels were normal. The patient tolerated the treatment well. The frequency of respiratory problems was reduced and the decline in the forced expiratory volume in 1 s and vital capacity reduced compared with the 4 yr preceding carrier erythrocyte therapy. Carrier erythrocyte-ADA therapy in an adult patient with ADA deficiency was shown to be metabolically and clinically effective.

The mouse immune response to carrier erythrocyte entrapped antigens.

This study investigated the potential of a single administration of carrier erythrocyte entrapped antigen to elicit humoral responses in the Balb/c mouse. Humoral responses to primary immunizations of erythrocyte encapsulated antigens were compared with those obtained with adjuvanted antigen administered via the subcutaneous route. Ig isotype responses to primary immunizations of erythrocyte entrapped antigen and subcutaneous antigen were compared to responses observed in mice that subsequently received booster immunizations with un-entrapped antigen. This study demonstrates that a single administration of antigen-loaded carrier erythrocytes is able to elicit humoral immune responses comparable or superior to those obtained via the adjuvanted subcutaneous vaccination route. The IgG isotype profiles demonstrate that the erythrocyte entrapment of antigens is another mechanism by which the Th responses to antigens maybe modulated.

Natural history, outcome, and treatment efficacy in children and adults with glutaryl-CoA dehydrogenase deficiency.

Glutaryl-CoA dehydrogenase (GCDH) deficiency is a rare inborn disorder of L-lysine, L-hydroxylysine, and L-tryptophan metabolism complicated by striatal damage during acute encephalopathic crises. Three decades after its description, the natural history and how to treat this disorder are still incompletely understood. To study which variables influenced the outcome, we conducted an international cross-sectional study in 35 metabolic centers. Our main outcome measures were onset and neurologic sequelae of acute encephalopathic crises. A total of 279 patients (160 male, 119 female) were included who were diagnosed clinically after clinical presentation (n = 218) or presymptomatically by neonatal screening (n = 23), high-risk screening (n = 24), or macrocephaly (n = 14). Most symptomatic patients (n = 185) had encephalopathic crises, characteristically resulting in bilateral striatal damage and dystonia, secondary complications, and reduced life expectancy. First crises usually occurred during infancy (95% by age 2 y); the oldest age at which a repeat crisis was reported was 70 mo. In a few patients, neurologic disease developed without a reported crisis. Differences in the diagnostic criteria and therapeutic protocols for patients with GCDH deficiency resulted in a huge variability in the outcome worldwide. Recursive partitioning demonstrated that timely diagnosis in neurologically asymptomatic patients followed by treatment with L-carnitine and a lysine-restricted diet was the best predictor of good outcome, whereas treatment efficacy was low in patients diagnosed after the onset of neurologic disease. Notably, the biochemical phenotype did not predict the clinical phenotype. Our study proves GCDH deficiency to be a treatable disorder and a good candidate for neonatal screening.

CFSUM1 and CFSUM2 in urine from patients with chronic fatigue syndrome are methodological artefacts.

McGregor et al. reported increased levels of an unidentified urinary compound (CFSUM1) in patients with chronic fatigue syndrome (CFS), with reduced excretion of another unidentified compound (CFSUM2), and suggested the possibility of chemical or metabolic 'markers' for CFS. The identity of CFSUM1 as reported was erroneous and the identities of these compounds have remained unknown until now. Urine samples were obtained from 30 patients with ME/CFS, 30 age- and sex-matched healthy controls, 20 control patients with depression and 22 control patients with rheumatoid arthritis. Samples were prepared using the published methods of McGregor et al. to produce heptafluorobutyryl-isobutyl derivatives of urinary metabolites. Alternative preparations utilised isopropyl, n-butyl and trifluoroacetyl derivatives. These were separated and identified using gas chromatography-mass spectrometry. CFSUM2 was identified as being partially derivatised [isobutyl ester-mono-heptafluorobutyryl (HFB)] serine. CFSUM1 was identified as partially derivatised pyroglutamic acid, being the isobutyl ester without formation of a HFB derivative. Both CFSUM1 and CFSUM2 are artefacts of the sample preparation procedure and previously reported quantitative abnormalities of CFSUM1 and CFSUM2 in urine from patients with ME/CFS are also artefactual. Pyroglutamic acid may be of primarily dietary origin. The methods used cannot provide reliable qualitative or quantitative data on urinary metabolites. No clinical or biochemical significance can be drawn between these compounds in ME/CFS or any other clinical conditions.

Riboflavin-responsive glutaryl CoA dehydrogenase deficiency.

We report here riboflavin responsiveness in a patient with glutaryl CoA dehydrogenase (GCDH) deficiency, compound heterozygous for the S139L and P248L mutations and with 20% residual GCDH enzyme activity in vitro. Our results suggest the mitochondrial GCDH homotetramer remains intact with one of these mutations associated with the binding site of the single FAD cofactor and that pharmacological doses of the cofactor precursor may be sufficient to induce an increase in activity in the mutant GCDH enzyme, although not sufficient to normalise urinary organic acid excretion. Serine139 is one of nine conserved amino acid residues that line the binding site of the protein and is in close proximity to both substrate and FAD cofactor. It is possible that steric alterations caused by substitution of serine with leucine at this position may be overcome with high cofactor concentrations. P248L is also associated with some residual GCDH activity in other patients and the unique combination of S139L with P248L may also explain the results in our patient. Responsiveness to riboflavin in our patient has been compared with two other patients with glutaric aciduria type 1 and minimal residual GCDH activity, one with homozygosity for the R257Q mutation and one with heterozygosity for the G354S mutation and a novel G156V mutation. A low lysine diet reduced glutaric acid excretion in our riboflavin-responsive GCDH-deficient patient almost to control values. She is now 21 years of age and clinically and neurologically normal.

Haemorheology in Gaucher disease.

UNLABELLED: In Gaucher disease, a deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within the lysosomes of the monocyte-macrophage system. Prior to the availability of enzyme replacement therapy (ERT), splenectomy was often indicated for hypersplenism. Haemorheological abnormalities could be expected in view of the anaemia and abnormal lipid metabolism in these patients and the role of the spleen in controlling erythrocyte quality. OBJECTIVES: To investigate the effect of Gaucher disease on blood and plasma viscosity, erythrocyte aggregation and erythrocyte deformability, and to determine whether observed rheological differences could be attributed to splenectomy. METHODS: Haematological and haemorheological measurements were made on blood collected from 26 spleen-intact patients with Gaucher disease, 16 splenectomised patients with Gaucher disease, 6 otherwise healthy asplenic non-Gaucher disease subjects and 15 healthy controls. RESULTS: No haemorheological differences could be demonstrated between spleen-intact patients with Gaucher disease and the control group. Compared to controls, both asplenic Gaucher disease and asplenic non-Gaucher disease study groups had a reduced MCHC (P = 0.003 and 0.005, respectively) and increased whole blood viscosity at 45% haematocrit (Hct), relative viscosity and red cell aggregation index - all measured at low shear (P < 0.05 for all). Additionally, asplenic patients with Gaucher disease alone showed an increased MCV (P = 0.006), an increased whole blood viscosity at 45% Hct measured at high shear (P = 0.019), and a reduced relative filtration rate (P = 0.0001), compared to controls. CONCLUSION: These observations demonstrate a direct and measurable haemorheological abnormality in Gaucher disease only revealed when there is no functioning spleen to control erythrocyte quality.

Urinary and plasma organic acids and amino acids in chronic fatigue syndrome.

Previous work by others have suggested the occurrence of one or more chemical or metabolic 'markers' for ME/CFS including specific amino acids and organic acids and a number of unidentified compounds (CFSUM1, CFSUM2). We have shown elsewhere that CFSUM1 is partially derivatised pyroglutamic acid and CFSUM2 partially derivatised serine and have suggested and demonstrated that the analytical methods used were unsuitable to identify or to accurately quantify urinary metabolites. We have now made a detailed analysis of plasma and urinary amino acids and of urinary organic acids from patients with ME/CFS and from three control groups. Fasting blood plasma and timed urine samples were obtained from 31 patients with CFS, 31 age and sex-matched healthy controls, 15 patients with depression and 22 patients with rheumatoid arthritis. Plasma and urinary amino acids and urinary organic acids were determined using established and validated methods and data compared by statistical analysis. None of the previously reported abnormalities in urinary amino acids or of organic acids could be confirmed. Results however provide some evidence in patients with ME/CFS for underlying inflammatory disease and for reduced intramuscular collagen with a lowered threshold for muscle micro-injury. These factors in combination may provide a basis for the fatigue and muscle pain that are the major symptoms in these patients.

Plasma and urinary carnitine and acylcarnitines in chronic fatigue syndrome.

Contradictory reports have suggested that serum free carnitine and acylcarnitine concentrations are decreased in patients with chronic fatigue syndrome (CFS) and that this is a cause of the muscle fatigue observed in these patients. Others have shown normal serum free carnitine and acylcarnitines in similar patients. We report here studies on free, total and esterified (acyl) carnitines in urine and blood plasma from UK patients with CFS and three control groups. Plasma and timed urine samples were obtained from 31 patients with CFS, 31 healthy controls, 15 patients with depression and 22 patients with rheumatoid arthritis. Samples were analysed using an established radioenzymatic procedure for total, free and esterified (acyl) carnitine. There were no significant differences in plasma or urinary total, free or esterified (acyl) carnitine between UK patients with CFS and the control groups or in renal excretion rates of these compounds. The data presented here show that, in the CFS patients studied, there are no significant abnormalities of free or esterified (acyl) carnitine. It is thus unlikely that abnormalities in carnitine homeostasis have any significant role in the aetiology of their chronic fatigue.

Methylmalonic aciduria unresponsive to vitamin B12: description of two cases and prenatal diagnosis in a brazilian family

Metilmalonicacidúria secundária à deficiência da mutase da metilmalonil CoA foi diagnosticada em duas crianças filhas de pais näo consangüíneos. A primeira criança morreu um dia antes que o diagnóstico correto fosse feito. Nesse período, sua mäe estava no terceiro trimestre de gestaçäo. Os níveis urinários de ácido metilmalônico foram medidos ao longo do terceiro trimestre de gestaçäo, mostrando um aumento gradual e indicando a presença de um feto homozigoto para a doença. A presença de metilmalonicacidúria foi confirmada no recém-nascido e o tratamento pode ser começado precocemente. A criança está sendo tratada com uma dieta com pouca proteína, com suplementaçäo de uma mistura de aminoácidos (exceto treonina, metionina, valina e isoleucina), DL-carnitina e bicarbonato. Está no momento fisicamente e mentalmente normal com doze meses de idade