Homozygosity for aquaporin 7 G264V in three unrelated children with hyperglyceroluria and a mild platelet secretion defect.

PURPOSE: Aquaporin 7 (AQP7) belongs to the aquaglyceroporin family, which transports glycerol and water. AQP7-deficient mice develop obesity, insulin resistance, and hyperglyceroluria. However, AQP7's pathophysiologic role in humans is not yet known. METHODS: Three children with psychomotor retardation and hyperglyceroluria were screened for AQP7 mutations. The children were from unrelated families. Urine and plasma glycerol levels were measured using a three-step enzymatic approach. Platelet morphology and function were studied using electron microscopy, aggregations, and adenosine triphosphate (ATP) secretion tests. RESULTS: The index patients were homozygous for AQP7 G264V, which has previously been shown to inhibit transport of glycerol in Xenopus oocytes. We also detected a subclinical platelet secretion defect with reduced ATP secretion, and the absence of a secondary aggregation wave after epinephrine stimulation. Electron microscopy revealed round platelets with centrally located granules. Immunostaining showed AQP7 colocalization, with dense granules that seemed to be released after strong platelet activation. Healthy relatives of these patients, who were homozygous (not heterozygous) for G264V, also had hyperglyceroluria and platelet granule abnormalities. CONCLUSION: The discovery of an association between urine glycerol loss and a platelet secretion defect is a novel one, and our findings imply the involvement of AQPs in platelet secretion. Additional studies are needed to define whether AQP7 G264V is also a risk factor for mental disability.

Homocysteine measurement by Vitros Microtip homocysteine assay.

BACKGROUND: Elevated circulating total homocysteine is an independent vascular risk factor. Enzymatic homocysteine measurements represent an alternative to HPLC- or immunochemistry-based assays, suitable for automation. Here, we report on analytical performance of a commercial cystathionine beta-synthase-based assay, for use on Vitros automated analyzers. METHODS: Linear range, limit of detection and analytical sensitivity were inferred from duplicate measurements of homocystine standard solutions (1-65 micromol/L). Imprecision was assessed using commercial controls according to NCCLS EP5-A2 and accuracy using NIST-SRM1955 reference material. Agreement with a clinically validated HPLC method was examined on 207 patient samples. RESULTS: The enzymatic assay was linear from 1 to 90 micromol/L homocysteine. Total (within-day) imprecision ranged from 4.5 (3.9)% to 2.8 (1.6)% at homocysteine 9.7-43.2 micromol/L. Accuracy was acceptable at 8.9 and 17.7 micromol/L homocysteine, with +6.4% and -1.2% bias, respectively, but showed substantial negative bias (-20.1%) at 4.0 micromol/L. High triglycerides (19.8 micromol/L) negatively interfered. The enzymatic method was slightly less sensitive than the HPLC method (limit of detection 0.7 and 0.2 micromol/L, respectively) but correlated well with the latter (r2=0.9997, slope=1.04, intercept=-0.66 micromol/L) and was more precise (p<0.05). CONCLUSIONS: The Vitros homocysteine assay met the CLIA Desirable Analytical Quality Specifications at homocysteine > or = 9 micromol/L. Its analytical performance and suitability for automation make the Vitros assay an analytically acceptable alternative to HPLC-based methods.

NMR spectroscopy of aminoacylase 1 deficiency, a novel inborn error of metabolism.

Aminoacylase 1 deficiency is a novel inborn error of metabolism. The clinical significance of the deficiency is under discussion, as well as the possible consequences of the defect for brain metabolism and function. This study includes the five originally published cases as well as three novel ones. NMR spectroscopy of urine, serum and cerebrospinal fluid has been used to study these patients. A typical profile with 11 accumulating N-acetylated amino acids was observed in urine from the patients. The concentration of most of the accumulating metabolites is typically 100-500 micromol/mmol creatinine. Two additional minor N-acetylated metabolites remain unidentified. The concentrations of the accumulating metabolites are <20 micromol/L in serum from the patients. Interestingly we found no evidence of an increased concentration of N-acetylated amino acids in the cerebrospinal fluid from one patient. Our data define aminoacylase 1 deficiency at the metabolite level providing a specific urinary profile of accumulating N-acetylated amino acids.

The aerobic forearm exercise test, a non-invasive tool to screen for mitochondrial disorders.

The diagnosis of a mitochondrial disorder is often difficult. Therefore, new approaches and diagnostic criteria are being developed. One of these tests is the aerobic forearm exercise test, a screening tool that can contribute to assess whether or not the patient suffers from a mitochondrial myopathy. With this simple, non-invasive test, the oxidative metabolism of muscle can be evaluated in rest and during exercise. We performed the aerobic forearm exercise test in patients with a mitochondrial disorder and an identified pathogenic gene mutation, in patients with a suspected mitochondrial disorder based on their clinical presentation and biochemical results, but without a molecular diagnosis, and in patients with atypical fatigue and no characteristics of a mitochondrial myopathy. In the first two groups, abnormal oxygen extraction from the blood during exercise was observed in four out of twelve patients. In the third group no abnormalities were found. The number of patients that we could test so far was limited, but all the patients experienced the aerobic forearm exercise as an easy test. We would like to stimulate clinicians to perform this test whenever a mitochondrial myopathy is suspected, as it can be a valuable diagnostic screening tool.

Plasma-equivalent glucose at the point-of-care: evaluation of Roche Accu-Chek Inform and Abbott Precision PCx glucose meters.

BACKGROUND: Glucose testing at the bedside has become an integral part of the management strategy in diabetes and of the careful maintenance of normoglycemia in all patients in intensive care units. We evaluated two point-of-care glucometers for the determination of plasma-equivalent blood glucose. METHODS: The Precision PCx and the Accu-Chek Inform glucometers were evaluated. Imprecision and bias relative to the Vitros 950 system were determined using protocols of the Clinical Laboratory Standards Institute (CLSI). The effects of low, normal, and high hematocrit levels were investigated. Interference by maltose was also studied. RESULTS: Within-run precision for both instruments ranged from 2-5%. Total imprecision was less than 5% except for the Accu-Chek Inform at the low level (2.9 mmol/L). Both instruments correlated well with the comparison instrument and showed excellent recovery and linearity. Both systems reported at least 95% of their values within zone A of the Clarke Error Grid, and both fulfilled the CLSI quality criteria. The more stringent goals of the American Diabetes Association, however, were not reached. Both systems showed negative bias at high hematocrit levels. Maltose interfered with the glucose measurements on the Accu-Chek Inform but not on the Precision PCx. CONCLUSIONS: Both systems showed satisfactory imprecision and were reliable in reporting plasma-equivalent glucose concentrations. The most stringent performance goals were however not met.

Preliminary performance evaluation of blood gas analyzers.

BACKGROUND: We evaluated the imprecision and bias of three instruments for the determination of blood gases, pH and ionized calcium (Ca(2+)) in human arterial blood samples, in comparison with the performance of an established methodology. METHODS: The ABL 735, Omni S and Rapidpoint 405 blood gas analyzers were evaluated and compared to the ABL 620 analyzer. Imprecision was determined according to the NCCLS EP10-A2 evaluation protocol. The NCCLS EP9-A2 evaluation protocol was used to determine bias relative to the ABL 620 system. Experimental data were compared against preset quality specifications. RESULTS: The three new instruments showed excellent imprecision for the measurement of pH, but only the ABL 620 met the preset imprecision goals for all analytes tested. All new instruments showed good correlation with the comparative instrument. The slope of the regression equation was significantly different from 1.0 in six out of the 12 comparisons, indicating systematic differences between the instruments. Nevertheless, the predicted bias values relative to the comparative instrument did not exceed the preset quality specifications for two out of the three new instruments. CONCLUSIONS: Preliminary evaluation using the NCCLS evaluation protocols EP10-A2 and EP9-A2, may provide valuable information on performance characteristics of blood gas analyzers.

Gas chromatographic-mass spectrometric analysis of N-acetylated amino acids: the first case of aminoacylase I deficiency.

During the metabolic work-up of a patient presenting with neonatal convulsions, we consistently observed the presence of unusual peaks in the gas chromatographic-mass spectrometric analysis of urinary organic acids. The gas chromatographic-mass spectrometric characteristics of the unusual peaks suggested that they corresponded to derivatives of N-acetylated amino acids. The tentative identification was confirmed by the identity of retention times and mass spectra of the trimethylsilyl derivatives of the authentic compounds. We describe our observations that led to the identification of the various N-acetylated amino acids in this first patient with a confirmed deficiency of aminoacylase I, an enzyme involved in the cytosolic degradation of N-terminally modified proteins. The potential and limitations of urinary organic acid analysis for the detection of N-acetylated amino acids was further studied using pure compounds. In addition, we provide mass spectral data for 37 trimethylsilyl derivatives from 17 N-acetylated amino acids, most of which have not been reported previously. Our data provide valuable information that will help the clinical laboratorians who are responsible for organic acid analysis to recognize this new condition and could aid its detection in other patients.

Clinical and diagnostic characteristics of complex III deficiency due to mutations in the BCS1L gene.

We investigated two siblings of a Spanish family presenting with congenital lactic acidosis. They had severe failure to thrive, liver dysfunction, and renal tubulopathy. An isolated biochemical complex III deficiency was detected in liver. A search for mutations in the human bc1 synthesis like (BCS1L) gene was undertaken. Direct sequencing revealed a missense mutation R45C and a nonsense mutation R56X, both located in exon 1 of BCS1L. The missense mutation in combination with a loss of function of the second allele is responsible for the isolated complex III deficiency in this family.