Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection.

Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20 min. The dynamic range of each amino acid was generally 1-1000 micromol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88-125%. Deming regression with the Beckman 7300 yielded slopes from 0.4-1.2, intercepts from -21 to 65 micromol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 micromol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, beta-alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors.

Relation of age, severity of illness, and hemodynamics with brain natriuretic peptide levels in patients <20 years of age with heart disease.

Brain natriuretic peptide (BNP) levels were obtained before cardiac catheterization in 193 pediatric patients with a variety of cardiac lesions. Age and functional status had strong relations to BNP values, with elevations of BNP levels associated with increasing functional disability and decreasing age. Mild but statistically significant correlations were found between BNP levels and right-sided cardiac pressures. In patients with volume-overloaded ventricles, BNP correlated with the degree of overcirculation.

Amino acid clearance during acute metabolic decompensation in maple syrup urine disease treated with continuous venovenous hemodialysis with filtration.

OBJECTIVE: Assessment of amino acid clearances by continuous venovenous hemodialysis with filtration in treatment of a metabolic decompensation in acute maple syrup urine disease. DESIGN: Single patient assessment. SETTING: Pediatric intensive care unit. PATIENTS: A 10-yr-old male with known maple syrup urine disease (branched chain alpha-ketoacid dehydrogenase deficiency) with metabolic decompensation due to an acute viral illness, characterized by altered mental status, progressive obtundation, and severe acidosis. INTERVENTIONS: Continuous venovenous hemodialysis with filtration. MEASUREMENTS AND MAIN RESULTS: Continuous venovenous hemodialysis with filtration was instituted with both filtration (500 mL/m(2)/hr) and dialysis (1000 mL/m(2)/hr) utilized, allowing rapid correction of systemic ketoacidosis while providing amino acid clearance. Amino acid clearance was measured at initiation and at 24 hrs into therapy. The procedure was well tolerated, with near normal mental status within 12 hrs and resumption of enteral feedings. During the 24-hr period of continuous venovenous hemodialysis with filtration, serum leucine levels fell from 2352 to 381 micromoles/L, isoleucine fell from 626 to 164, and valine fell from 1117 to 228. Leucine, isoleucine, and valine clearance rates averaged 13.1, 12.8, and 13.2 mL/min, respectively, and were constant during the 24 hrs of treatment. Clearance of other amino acids during this period did not vary significantly between cationic, anionic, neutral, or hydrophobic amino acids. CONCLUSIONS: Continuous venovenous hemodialysis with filtration provides an effective therapeutic alternative to intermittent hemodialysis during acute metabolic decompensation in maple syrup urine disease.

Analysis of cerebrospinal fluid glial fibrillary acidic protein after seizures in children.

PURPOSE: To evaluate pediatric seizure patients for astrocytic injury by measuring cerebrospinal fluid (CSF) glial fibrillary acidic protein (GFAP), determine risk factors for GFAP elevation after seizures, and compare seizure-induced astrocyte injury with neuronal injury by concurrent measurement of CSF neuron-specific enolase (NSE). METHODS: CSF obtained from pediatric patients (n = 52) within 24 h of seizure was assayed for GFAP and NSE. Retrospective chart review was performed for seizure type, duration, and etiology. RESULTS: Overall, children with seizures had elevated CSF GFAP compared with controls (p = 0.0075), but no elevation of NSE (p = 0.1437). No effect of seizure type or etiology was found, but a significant positive effect of seizure duration (p = 0.0010) and status epilepticus (p = 0.0296) was seen on CSF GFAP. Individually, seven children (13%) had elevated GFAP (>440 pg/ml); in five children, the increased GFAP was not accompanied by elevations in NSE (<12 ng/ml). Five children with elevated GFAP had symptomatic etiologies for their seizures, but the etiology of one child with elevated GFAP was cryptogenic, and one had febrile seizures. CONCLUSIONS: Elevation of CSF GFAP after seizures suggests that astrocytic injury may occur in a subgroup of children, primarily in the context of prolonged seizures and symptomatic etiologies. Increased GFAP levels may occur in patients with normal NSE, suggesting that GFAP may be a more sensitive marker of brain injury in some cases.

Role of protein binding in renal elimination of leptin.

OBJECTIVE: Leptin, a hormone produced by fat which signals to the brain the extent of fat stores, is known to be eliminated from circulation primarily by the kidney. The hormone circulates in both free and protein-bound forms, but there is little information concerning the inter-relationship of these forms of leptin, or which form is influenced by physiological processes such as renal elimination. We studied total, free and bound concentrations of leptin in ambulatory adults undergoing catheterization for diagnosis/management of congenital cardiac disease. DESIGN: Blood specimens were collected from both the arterial circulation and the renal vein, for determination of the fractional extraction of leptin resulting from a single pass through the kidney. PATIENTS: Thirteen subjects were studied. MEASUREMENTS: Total leptin concentrations were measured by radioimmunoassay, and free/protein-bound leptin concentrations were quantified by an high-performance liquid chromatography method. Adequacy of renal vein sampling was assessed by comparing the creatinine concentration of arterial and venous specimens. RESULTS: Mean fractional extraction of creatinine was 28 +/- 7% in the 13 subjects studied. Fractional extraction of total leptin was 18 +/- 8%, significantly less than that for creatinine. Fractional extraction of total leptin was not related to arterial leptin concentration or the fractional extraction of creatinine. Both free and bound fractions of leptin were significantly reduced by passage through the kidney, with fractional extractions of 22% and 25%, respectively. Efficiency of extraction was not influenced by the relative proportion of free or bound leptin fractions. Leptin-binding capacity (a measure of the concentrations of leptin-binding proteins) was not altered by passage through the kidney. CONCLUSIONS: Both free and bound leptin are metabolically active with regard to elimination. Protein-bound leptin equilibrates with the free leptin fraction in circulation as the result of a dynamic equilibrium. The data are consistent with either glomerular filtration or active uptake as mechanisms of elimination. Leptin-binding proteins are apparently neither eliminated or produced by the kidney.

Whole blood international normalization ratio measurements in children using near-patient monitors.

PURPOSE: To report a comparison of international normalization ratio (INR) measurements on four near-patient (point-of-care or bedside) whole blood INR monitors in children. PATIENTS AND METHODS: The INR results from 19 ambulatory pediatric subjects (30 hospital visits) receiving warfarin sodium were analyzed on four near-patient monitors and compared with plasma INR measurements on the laboratory CA-1000 Analyze. The instruments evaluated were CoaguChek, Hemochron Jr. Signature, ProTime Microcoagulation System, and RapidpointCoag. RESULTS: The INR measurements ranged from 1.05 to 5.25. Over the entire INR range, the near-patient instrument with the least bias relative to the CA-1000 was the RapidpointCoag (r(2) = 0.923). The correlations (r(2)) of the CoaguCheck, Hemochron Jr., and ProTime were 0.877, 0.834, and 0.885, respectively. Precision studies involved repeated analysis of one nonmedicated adult (mean CA-1000 INR = 0.908) and one adult receiving oral anticoagulation therapy (mean CA-1000 INR = 2.42). The coefficient of variation on the near-patient monitors for both adult volunteers ranged from 4.9% to 22.3%. Bilirubin levels up to 20 mg/dL did not interfere in any of the methods. CONCLUSIONS: Near-patient testing whole blood INR monitors offer acceptably accurate and precise measurements. Values obtained on near-patient monitors may vary considerably from the reference method, and data obtained should serve as a supplement to, but not a replacement for, routine clinical laboratory measurements.

Home sodium monitoring in patients with diabetes insipidus.

OBJECTIVE: To determine whether home care givers can accurately measure plasma sodium in children with diabetes insipidus (DI) by using an I-STAT portable clinical analyzer (PCA) and to collect preliminary data on home PCA use. STUDY DESIGN: Care givers of 4 children with DI and impaired thirst or inability to access water freely were instructed in PCA use. During an initial preclinical phase, the accuracy of sodium concentration measured by care givers was assessed by comparison to simultaneous analysis in a clinical laboratory. Participants were subsequently randomly assigned to daily home PCA monitoring or routine care. All participants crossed over from their original randomized group assignment to the alternate group. RESULTS: After a single education session, all care givers were able to perform PCA testing. There was good correlation between PCA and laboratory sodium (r = 0.92). On the basis of Error Grid Analysis, use of the PCA sodium would have resulted in treatment decisions identical to those made based on the laboratory sodium value in 62 of 66 instances. Four minor differences in treatment would have occurred. There was no statistically significant difference in clinical outcome during daily monitoring versus routine care. CONCLUSIONS: Results obtained by care givers using the PCA are sufficiently reliable for assessment of fluid status and making treatment decisions.

Macro-aspartate aminotransferase in a female with antibodies to hepatitis C virus.

Persistent elevation of aspartate aminotransferase (AST) activity in serum due to the presence of a macroenzyme form of AST (macro-AST) may lead to diagnostic confusion in many clinical conditions, particularly those associated with chronic liver disease. We describe a case of macro-AST arising in an adult female with a false-positive hepatitis C virus (HCV) RNA test result that was not accompanied by other biochemical or histologic evidence of liver disease. The presence of macro-AST in serum was confirmed utilizing size-exclusion, high performance liquid chromatography (HPLC) and Protein G-agarose beads to precipitate immune complexes of AST and immunoglobulin G followed by centrifugation and AST activity measurements in the supernatant. A brief review of the clinical enzymology of AST and methods used to quantify serum macro-AST activity is provided.

Accurate direct spectrophotometric bilirubin measurement combined with blood gas analysis.

BACKGROUND: A new total bilirubin (B(T)) method, based on multiple wavelength absorbance measurements, and an algorithm to calculate concentration, were evaluated for accuracy in specimens containing variable amounts of unconjugated bilirubin (B(U)), conjugated bilirubin (B(C)) and delta (protein-bound) bilirubin (B(D)). METHODS: Quantitation of B(U), B(C), and B(T) (with calculation of B(D)) using a Vitros 250 analyzer served as the comparison method. RESULTS: Analysis of neonatal specimens using a preliminary algorithm yielded good overall agreement with the Vitros B(T) method, but there was considerable variation in the agreement for individual specimens. When specimens from adults selected to yield a range of B(C) and B(D) levels were analyzed, the preliminary algorithm underestimated B(T). Refinement of the method in the form of a finalized algorithm resulted in elimination of the negative bias seen with specimens with high B(D) and B(C) levels, and better agreement for individual neonatal specimens. CONCLUSIONS: This new method overcomes the limitations observed in earlier spectrophotometric methods, and provides accurate results in specimens containing a range of bilirubin forms.

Cerebrospinal fluid neuron-specific enolase following seizures in children: role of etiology.

Neuron-specific enolase, a marker for neuronal injury, is elevated following seizures in adults, but relatively few data exist on postictal neuron-specific enolase levels in children. This study measured cerebrospinal fluid (CSF) neuron-specific enolase levels after seizures in 49 consecutive pediatric patients and investigated the role of seizure type, duration, and etiology in influencing neuron-specific enolase. Overall, there was no significant difference in neuron-specific enolase levels between patients with seizures and a control group. However, 4 of the 49 seizure patients (8%) had neuron-specific enolase levels clearly above the normal range. Seizure patients with symptomatic etiologies had significantly increased neuron-specific enolase compared to cryptogenic/idiopathic or febrile seizures. The four individual patients with elevated cerebrospinal fluid neuron-specific enolase all had identified metabolic or genetic etiologies and presented with medically refractory status epilepticus. No individuals with cryptogenic/idiopathic or febrile seizures had abnormal neuron-specific enolase. There was no significant effect of seizure duration or type on cerebrospinal fluid neuron-specific enolase. In contrast to adults, acute seizure-induced neuronal injury in children as detected by neuron-specific enolase is rare and may occur primarily with severe symptomatic etiologies. Children with cryptogenic, idiopathic, or febrile seizures, including status epilepticus, are at relatively low risk for neuronal damage following seizures.

Variation in plasma leptin concentrations after unilateral nephrectomy.

The temporal changes in plasma leptin concentrations were studied in healthy adults who underwent unilateral nephrectomy. Another group who underwent abdominal surgery for repair of aneurysm or to relieve arterial stenosis, was also studied. Plasma leptin concentrations increased to 230% +/- 74% of prenephrectomy levels at 8 to 16 hours after surgery and then generally declined. Subjects with prenephrectomy leptin concentrations above 14 microL maintained elevated postnephrectomy levels, whereas subjects with low prenephrectomy concentrations had final leptin levels below prenephrectomy concentrations. Abdominal surgery subjects did not manifest the increase after surgery, but generally had declining concentrations throughout the convalescent period. Free and bound fractions of plasma leptin and leptin binding capacity were measured in the prenephrectomy and peak specimens (8 to 16 hours postnephrectomy) by high-performance liquid chromatography (HPLC). The increase in total leptin postnephrectomy largely affected the free fraction of leptin, without significant increase in bound leptin or leptin binding capacity. We conclude that (1) plasma leptin concentrations increase acutely after nephrectomy, consistent with the role of the kidneys in eliminating circulating leptin; (2) plasma leptin concentrations decline thereafter, suggesting activation of compensatory elimination capacity; and (3) the postnephrectomy peak in total leptin increases primarily free leptin.

Artifícios analíticos nas determinações do hematócrito por analisadores químicos em sangue total / Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers

Analisadores compactos, apropriados para testes em pacientes à mão, avaliam o hematócrito pela medida da condutividade do sangue não diluído. Nós avaliamos a exatidão do resultado de hematócrito de um determinado analisador (Instrumentation Laboratory BGE Analyzer) em comparação com um automático contador eletrônico de partículas (CEP) e volume de células sedimentadas (VCS) microhematócrito. Quando amostras (n = 34) de pacientes externos e de enfermaria foram analisadas por todos os três métodos, o analisador AEG estava de acordo com os dois hematócritos CEP e VCS (AEG = 1,00 VCS + 0,3%, Sy/x = 1,6% ; AEG = 1,04 CEP + 0,4%, Sy/x = 2,0%), indicando que todos os três métodos têm performances similares na maioria dos pacientes. Entretanto, um paciente com osmolalidade plasmática aumentada mostrou significante decréscimo nos hematócritos AEG e VCS em comparação ao método CEP. As diferenças nas medidas do hematócrito podiam ser reproduzidas pela adição de solutos ao sangue “in vitro” ou pela modificação de osmolalidade plasmática de ratos "in vivo". Amostras de pacientes submetidos a uma cirurgia cardíaca, cujo sangue tinha grandes mudanças na concentração de proteína, mostrou discrepância entre hematócritos pela condutividade e outros métodos; efeitos similares poderiam ser produzidos pelas mudanças na concentração de proteína ou pela adição "in vitro" de polietileno glicol. Nós concluímos que medidas de condutividade fornecem resultados exatos do hematócrito para sujeitos normais fisiologicamente, mas não para alguns pacientes sob cuidado intensivo e pacientes cirúrgicos.