Normal Control Values Research Articles

Increased serum placenta growth factor level is significantly associated with progression, recurrence and poor prognosis of oral squamous cell carcinoma

We recently found that the expression of placenta growth factor (PlGF) in oral squamous cell carcinoma (OSCC) specimens is correlated with the progression and prognosis of OSCC. In this study, serum samples were obtained from 72 OSCC patients before and 3 months after surgical cancer excision and from 30 normal controls. Serum PlGF levels were determined by enzyme-linked immunosorbent assay (ELISA). The mean serum PlGF levels were significantly higher in pre-surgery OSCC patients than in normal controls (19.1±10.7 vs. 10.1±4.5, P<0.001). Serum PlGF levels dropped to near the normal control levels after surgical cancer removal. Higher pre-surgery serum PlGF levels were significantly associated with larger tumor size (P=0.015), positive lymph node metastasis (P=0.001), more advanced clinical stages (P=0.002), and loco-regional recurrence (P=0.037). The serum PlGF level was identified as an independent unfavorable prognosis factor by multivariate Cox regression analyses (P=0.014). Kaplan-Meier curve showed that OSCC patients with a higher serum PlGF level had a significantly poorer cumulative recurrence-free survival than those with a lower serum PlGF level (log-rank test, P=0.009). When we used the serum PlGF level of 19.1 pg/ml (mean normal control value plus 2 standard deviations) as a cutoff point, the sensitivity, specificity, and positive predictive value for tumor recurrence was 80%, 56% and 78%, respectively. We conclude that the serum PlGF level may be a valuable biomarker for prediction of therapeutic effect, progression, recurrence and prognosis of OSCC.

Effect of New Direct Oral Anticoagulants on Ex Vivo Platelet and Fibrin Thrombus Formation in Blood Flowing Over Fibrillar Type I Collagen

Abstract 2318 Background.Studies have shown that 40–85% of patients undergoing total knee replacement develop venographically confirmed deep vein thrombosis (DVT) if not given post-operative thromboprophylaxis; approximately 0.1 to 1.7% of these patients will suffer fatal pulmonary embolism (PE). Oral anti-vitamin K anticoagulants are effective for the prevention and treatment of venous thrombosis, but have limitations. In particular, they have multiple food and drug interactions as well as variable pharmacokinetics and pharmacodynamics, such that regular laboratory monitoring and dose adjustments are required to maintain an optimal therapeutic range as defined with the International Normalized Ratio (INR). New oral agents that inhibit coagulation factor Xa or thrombin have been developed and shown to be effective and safe without requiring laboratory monitoring. In view of the relevance of the latter point, we have studied patients treated with an oral anti factor Xa agent (Rivaroxaban) or Coumadin, and evaluated the antithrombotic efficacy of the respective drugs by measuring platelet aggregation and fibrin deposition in patient blood perfused over fibrillar collagen type I. Material and Methods. Blood drawn from an antecubital vein and containing 0.011 M trisodium citrate as anticoagulant was recalcified with 5 mM calcium chloride and immediately perfused through a rectangular chamber mounted on the stage of a confocal microscope and presenting a surface coated with fibrillar collagen type I under laminar flow conditions at the wall shear rate of 300 1/s. Platelets and fibrin were specifically detected in situ through distinct fluorochromes. We tested 8 normal controls, 8 patients treated with Coumadin and a stable INR value between 1.94 and 2.90 (mean 2.34; standard deviation 0.34), and 7 patients treated with Rivaroxaban at between 8 and 16 days (mean 12.14; standard deviation 2.48 days) from the initiation of therapy. The volume of platelet aggregates and fibrin deposited onto the collagen fibrils was measured distinctly from stacks of confocal sections by integrating surface coverage of each thrombus component in consecutive optical planes separated by 2 micrometers in height. Results and Discussion. There was no significant difference in the volume of platelet and fibrin aggregates formed in blood of normal control and patients treated with either Coumadin or Rivaroxaban. This result was surprising because the patients treated with Coumadin had a laboratory demonstration of significantly retarded coagulation. We reasoned, however, that coagulation tests are typically performed in platelet-poor plasma, while in the perfusion assay coagulation occurs in whole blood and on a surface onto which flowing platelets are fully activated, thus increasing the local procoagulant potential. For this reason, we performed a series of experiments in which a variable amount of a highly specific thrombin inhibitor, lepirudin, was titrated into the recalcified blood before perfusion. We thus determined that with 50 nM lepirudin added to blood there was no decrease in the volume of platelet aggregates and fibrin deposited onto collagen in blood of normal individuals, while the volume of fibrin was decreased in patients receiving either Coumadin or Rivaroxaban. The corresponding values for normal controls, Coumadin-treated and Rivaroxaban-treated patients, in the order, were (mean volume ± standard error of the mean in cubic micrometers): Platelet aggregates = 28,592±3,354; 36,959±4,973; 44,448±7,110; Fibrin = 84,190±9,740; 47,298±7,308; 35,780±5,091. The differences in platelet aggregate volumes were not significant, while fibrin volume was significantly smaller in the anticoagulant-treated patients as compared to normal (p<0.01 for Coumadin and p<0.001 for Rivaroxaban); the difference between patients treated with one or the other anticoagulant was not significant. These results show that Rivaroxaban and Coumadin at therapeutically effective dosage have comparable effect in reducing thrombin generation, as evidenced by the reduced volume of fibrin formed in flowing blood exposed to collagen. This, however, is accompanied by an increased volume of platelet aggregates on the highly thrombogenic collagen surface. The relevance of these experimental results with respect to prevention of arterial as opposed to venous thrombosis deserves further investigation. Disclosures:No relevant conflicts of interest to declare.

Age-related decrease of miRNA-92a levels in human CD8+ T-cells correlates with a reduction of naïve T lymphocytes

MicroRNA (miR)-17-92a expression plays a crucial role in lymphocyte ontogeny. We therefore set out to determine miR-92a expression levels in peripheral blood lymphocytes from healthy subjects to ascertain any association between these levels and ageing. We found a positive correlation between the miR-92a expression level and the percentages of RO-CD8+CD27+ (P = 0.0046) and CD3+CD8+CD62L+ (P = 0.0011). This suggests that the majority of miR-92a of CD8+ T cells is derived from naïve cells, and the miR-92a expression level in CD8+ T cells declines progressively with age. These results indicate that the age-related attrition of naïve T cells is linked to a reduction of miR-92a in human T -lymphocytes. Therefore, we should careful attention when evaluating human miRNA levels in T lymphocytes to use normal control values.

Heterozygous signal transducer and activator of transcription 3 mutations in hyper-IgE syndrome result in altered B-cell maturation

Heterozygous signal transducer and activator of transcription 3 mutations in hyper-IgE syndrome result in altered B-cell maturation

Biventricular Performance in Patients with Marfan Syndrome without Significant Valvular Disease: Comparison to Normal Subjects and Longitudinal Follow-Up

The presence and progressive nature of primary myocardial involvement in Marfan syndrome are debated. The aim of this study was to evaluate the clinical relevance of left ventricular (LV) and right ventricular (RV) strain in adult patients with Marfan syndrome without significant valvular disease. Adult patients with Marfan syndrome (n= 50; mean age, 35.2 ± 12.9 years) were followed prospectively. Echocardiography was performed annually and consisted of comprehensive assessment of ventricular and valvular function. Using speckle-tracking imaging, the baseline strain values of the Marfan population were calculated and compared with the values of normal controls. The follow-up evaluations were used to assess changes in ventricular strain. The association between the incidence of adverse events (heart failure, [supra]ventricular arrhythmias, and proximal aorta surgery) and baseline strain values was investigated. Compared with controls, patients with Marfan syndrome had significantly lower peak longitudinal LV strain (-18.9 ± 2.3% vs -20.1 ± 1.9%, P < .01) and RV strain (±26.9 ± 5.2% vs ±29.3 ± 4.25%, P < .01). The absolute changes in LV longitudinal, radial, and circumferential strain and RV longitudinal strain during a median 4 years of follow-up were 0.1 ± 2.8%, 1.12 ± 7.6%, 0.3 ± 3.7%, and 0.9 ± 5.5%, respectively, which was not statistically significant. Cox regression demonstrated that reduced LV or RV strain was not associated with adverse outcome (supraventricular arrhythmias, n= 3; proximal aorta surgery, n= 4). This study suggests that patients with Marfan syndrome show lower ventricular strain and strain rate values compared with healthy controls. However, no relevant changes in LV and RV function occurred during midterm follow-up in patients with Marfan syndrome without valvular disease at baseline. Although ventricular strain and strain rate were mildly reduced in patients with Marfan syndrome, this did not affect outcomes negatively in the present study.

Abdominal ultrasound with scintigraphic and clinical correlates in infants with sickle cell anemia: baseline data from the BABY HUG trial.

The purpose of this study is to perform and evaluate baseline abdominal ultrasound in infants with sickle cell anemia who participated in the BABY HUG multiinstitutional randomized placebo-controlled trial of hydroxyurea therapy and to examine the potential relationships among ultrasound results and clinical, nuclear medicine, and laboratory data. After local institutional review board approval and with informed guardian consent, 116 girls and 87 boys (age range, 7.5-18 months) with sickle cell anemia underwent standardized abdominal sonography at 14 institutions. Imaging was centrally reviewed by one radiologist who assessed and measured the spleen, kidneys, gallbladder, and common bile duct. Baseline physical assessment of spleen size, serum alanine aminotransferase and bilirubin levels, (99m)Tc sulfur colloid liver-spleen scans, and (99m)Tc diethylenetriaminepentaacetic acid clearance glomerular filtration rates (GFRs) were obtained. Analysis of variance and the Student test were performed to compare sonographic findings to published results in healthy children and to clinical and laboratory findings. The mean (± SD) spleen volume (108 ± 47 mL) was significantly greater than published normal control values (30 ± 14 mL; p < 0.0001). There was no correlation between spleen volume and function assessed by liver-spleen scan. The mean GFR (125 ± 34 mL/min/1.73 m(2)) was elevated compared with control GFRs (92 ± 18 mL/min/1.73 m(2)). Renal volumes (right kidney, 29 ± 8 mL; left kidney, 31 ± 9 mL) were significantly greater than control volumes (right kidney, 27 ± 3 mL; left kidney, 27 ± 3 mL; p < 0.0001) and were positively correlated with GFR (p = 0.0009). Five percent of patients had sonographic biliary abnormalities (sludge, n = 6; dilated common bile duct, n = 2; and cholelithiasis and thickened gallbladder wall, n = 1 each). There was no correlation between biliary sonographic findings and laboratory results. In infants with sickle cell anemia, sonographic spleen volume does not reflect function, but increased renal volume correlates with GFR and is consistent with hyperfiltration. Sonographic biliary abnormalities can occur early in life, while remaining clinically silent.

Possible mechanisms for N-acetyl cysteine and taurine in ameliorating acute renal failure induced by cisplatin in rats

Context: Cisplatin (CP), a broadly used anticancer drug, is widely known to induce acute renal failure as a result of renal tubular injury.Objective: In this study, the effect of N-acetyl cysteine (NAC) or taurine (TAU) for protection against CP-induced nephrotoxicity was investigated.Materials and methods: A single dose of CP 1 mg/kg b.wt. for 4 days was given IP to the rats, 10 days rest, and then the dose was repeated for other 4 days. After CP administration, NAC or TAU was given IP in a dose of 50 mg/kg b.wt. 3 times weekly for 8 weeks.Results: CP-induced nephrotoxicity is reflected in high values of blood urea and creatinine levels. In addition, the significant increase in the β2-MG and N-acetyl-β-glucosaminidase enzymes exhibited a strong correlation with histology scores of overall proximal tubule damage as compared with the normal control values. Treatment with TAU or NAC after CP administration significantly ameliorated CP-induced nephritic oxidation stress markers as compared with CP-treated group. On the other hand, treatment with TAU or NAC after CP administration significantly ameliorated CP-induced nephritic inflammation with possible attenuation of renal injury.Discussion: These data suggest that oxidative stress and inflammation are involved in the pathogenesis of CP-induced acute renal failure. The inhibition in oxidative stress and the elevation of the total antioxidant capacity as well as the inhibition of the inflammatory biomarkers by NAC or TAU after CP administration may play a central role in modulation of nephrotoxicity induced by CP.Conclusion: NAC and TAU have antioxidant and anti-inflammatory against CP-induced nephrotoxicity.

The protective effect of probucol on adriamycin nephrosis in the rat.

Recent research has indicated the role of reactive oxygen species (ROS) in experimental nephritis. We examined the role of ROS and the effect of probucol, an anti-hyperlipidemic drug with antioxidant activity, on adriamycin (ADR)-induced nephrosis in the rat. Fourteen days after single intravenous injection of ADR (7.5 mg/kg b.w.), a nephrotic state was observed. Compared with the normal control values, the total kidney glutathione content was lower on day 5, but significantly higher on day 14 in the ADR-injected rats. Feeding ADR-injected rats with food containing 1% probucol was effective in reducing urinary protein excretion. Serum lipid peroxide level and kidney total glutathione content, both of which increased on day 14 in the ADR-injected rats, were also decreased significantly by concomitant probucol treatment. During long-term observation period of 18 weeks, probucol treatment relieved both urinary protein excretion and the progression of renal impairment. These protective effects of probucol suggest a role of ROS in the induction and progression of ADR nephrosis.

Effect of glucomannan on haematological, coagulation and biochemical parameters in male rabbits fed aflatoxin-contaminated ration

The main goal of the present work was to explore the effect of glucomannan (GM) as a biological detoxifying agent on haematological, coagulation and biochemical parameters in male rabbits fed aflatoxin B1 (AFB1)-contaminated ration. Thirty male White New Zealand rabbits were used in this study. They were divided into 3 equal groups. The first group was given a ration free from mycotoxins and kept as a control group. Rabbits in the second group were fed a ration naturally contaminated with AFB1 (0.1 mg/kg ration) for 28 days, while rabbits in the third group were fed AFB1-contaminated ration (0.1 mg/kg ration) in addition to GM (1 kg/ton) for 28 days. The results of the present work revealed that aflatoxicosis elicited a picture of microcytic hypochromic anaemia, leukopenia, lymphocytopenia and monocytopenia. Coagulopathy recorded in this work due to aflatoxicosis was characterised by thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time. Hepatotoxicity due to AFB1 recorded in this work was characterised by a significant increase in serum activity of alanine aminotransferase, gammaglutamyl transferase and total bilirubin in addition to a significant decrease in serum total proteins and albumin. Data recorded in this work showed a significant increase in serum urea, creatinine, total lipids and cholesterol in addition to a significant decrease in serum glucose, calcium, phosphorus and iron in rabbits fed aflatoxin-contaminated ration only. Supplementation of rabbit ration with GM resulted in the return of the above-mentioned parameters to normal control values. It could be concluded that GM provides new insights into counteracting the adverse effects of AFB1.

Influence of Cell-Derived Microparticles on Thromboelastography.

AbstractAbstract 3698 Background.Thromboelastography (TEG) is a method of analyzing coagulation commonly used to assess pre- and perioperative bleeding risk. We have been interested in the role of cell-derived microparticles (MP) in coagulation and in this study, examined correlations between TEG parameters and MP levels as described in Methods. Methods.Patients: Whole citrated blood from a total of 43 patients was examined by TEG (Haemoscope TEG 5000), within 5 hours of drawing. The study subjects consisted of 12 ITP, 5 TTP, 26 thrombocytopenia (TP) of various causes and 21 healthy controls. TEG: Parameters analyzed were maximum amplitude (MA), clot strength (G), lag time (R), time to formation of clot of amplitude 20 mm (k), rate of growth (a), coagulation index (CI). MP Assay: MP from platelets (PMP) were defined flow cytometrically by CD31+/CD41+; from red cells (RMP) by glycophorin A (CD235); from endothelia (EMP) by CD62E; and from leukocytes (LMP) by CD45. In addition, we employed generic markers, lectin FITC-Ulex europaeus (Ulex) as a measure of “total MP”; and annexin V+ (AnV+) MP, a marker of procoagulant phospholipids. Platelet counts were obtained from CBC at the clinical laboratory. Results.We assessed correlations between each TEG parameter, and each MP parameter, as well as platelet counts. Only those which were significant (p<0.05) are reported below. (1) RMP levels correlated positively and strongly with lag time, R (r=0.737, p<0.0001). LMP levels correlated positively with k (r=0.553, p=0.0001) moderately. Interestingly, we found moderate but consistent positive correlation between PMP levels and MA, G, and CI. These values were r=0.314, r=0.317, and r=0.346, respectively, and p<0.05 in each case. (2) Normal control values had an average G of 7.86 ±1.70 Kd/cm2 (mean ±SD), in contrast to ITP, TTP, and TP patients which had average G's of 8.62 ±3.96, 10.34 ±1.97, and 5.96 ±3.98 Kd/cm2, respectively. However, no statistically significant differences of means between patient groups and controls were found for any parameter in these small populations. (3) Platelet count was found to have a moderate correlation with G in TP patients (r=0.427, p<0.05) and with k (r= -0.459, p=0.018), but neither variable (G, k) correlated with platelet counts in ITP or TTP, possibly owing to the smaller populations in those groups. (4) Examining all patients combined for correlation between platelet count and G, we found r=0.633 (p<0.0001), confirming previous studies suggesting a relation between platelet count and G in both healthy and diseased patients. High platelet count was reasonably well correlated to high PMP (r=0.506, p<0.001). This may indicate that platelet activation as well as platelet count influences TEG, as it has been shown that PMP levels are a proxy of platelet activation. Conclusions:We conclude that MP contributes significantly to multiple variables in TEG profiles of patients with ITP, TTP, and TP, some positively, some negatively; and that there is wide variability in MP levels among these diseases. Platelets clearly also modulate TEG parameters, although the interplay between platelets and MP in forming a stable clot bears further research. PMP have the largest array of effects, positively correlating with MA, G, and CI, and negatively correlating with R+k, while having no significant effect on the other parameters. The data suggest that nearly all the MP affect the TEG parameters significantly. The most commonly affected parameter was MA, which is correlated to EMP, LMP, PMP, and Ulex. G and CI were also frequently affected by various MPs. This study is limited by the small patient population, various medications, and varying degree of severity of diseases. Disclosures:No relevant conflicts of interest to declare.

Correlation between LV Regional Strain and LV Dyssynchrony Assessed by 2D STE in Patients with Different Levels of Diastolic Dysfunction

Little is known about left ventricular (LV) circumferential, radial, and longitudinal functions in patients with diastolic dysfunction (DD). The aims of this study were: (1) to characterize circumferential, radial, and longitudinal strain and (2) to use 2D speckle tracking echocardiography (STE) to evaluate LV dyssynchrony in different DD grades. We studied 128 subjects (90 with DD, 38 healthy controls). DD was divided into three groups according to grade (group 1; mild, group 2; moderate, and group 3; severe). Regional strains were measured by 2D STE. Global LV CS was significantly lower in group 3 (-17.4 ± 5.2) compared with healthy controls (-21.2 ± 3.6), group 1 (-20.7 ± 3.9), and group 2 (-20.2 ± 4.0) (P < 0.0001 for each comparison). Global LV RS and LS were significantly lower in all three groups than in controls. LV, LS, and RS were lower in patients with mild to severe DD than in healthy controls. However, LV CS was similar to normal control values in patients with mild to moderate DD but was lower in patients with severe DD. Circumferential segmental LV motion is likely to compensate for impaired longitudinal and radial LV motion in patients with DD. LV dyssynchrony also occurred significantly more frequently in patients with DD with normal EF than in healthy controls. These results suggest that, in patients with DD and normal EF, contraction of the myocardium is not synchronized and that LV dyssynchrony plays a role in producing diastolic heart failure.

Evaluation of Plasmodium vivax ELISA for the blood screen

Plasmodium vivax malaria is the indigenous strain in the Republic of Korea (ROK). Plasmodium vivax can be transmitted through the transfusions of various blood components, which became a severe problem with the safety of blood transfusions and blood-related products in ROK. We evaluated a P.vivax-specific enzyme-linked immunosorbent assay (Genedia Malaria Ab ELISA 2.0, Green Cross, ROK) with blood samples from four groups: 251 samples from P.vivax-infected patients, 39 samples from post-treatment patients upon follow-up, 200 samples from healthy volunteers and 421 samples from domestic travellers to and from high endemic areas of ROK. The positive cases from the ELISA test were confirmed by both Giemsa microscopic and polymerase chain reaction methods. The clinical sensitivity and specificity of detecting P.vivax with ELISA test were 94.4% and 99.0%, respectively. Thirteen of 421 domestic travellers (3.0%) to endemic areas tested positive. The results indicate the effectiveness of detecting antibodies against P.vivax in blood with Genedia Malaria Ab ELISA 2.0 test in a large blood screen setting.

E0545 Research on inhibiting vascular remodelling of Captopril and Xuefuzhuyu Capsule on the basis of restraining activation and migration of adventitial fibriblasts

BackgroundVascular remodelling is the common pathophysiological basis of artherosclerosis, hypertension and coronary artery disease, et al. It is also one of pathogenesis mechanisms which causes target organs damage and dysfunction....

Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies

The diagnosis of transferase and galactokinase deficiency galactosemia usually involves the measurement of erythrocyte galactose-1-phosphate uridylyltransferase (GALT) and galactokinase (GALK) enzyme activity, respectively. The current gold standard assays for these enzymes are radioactive assays, which are laborious and/or incapable of measuring low enzyme activities. To further our knowledge of genotype–phenotype relationships, we had developed an assay for GALT activity alone using LC–MS/MS. In this study we generated a robust and sensitive LC–MS/MS based GALT and GALK assay using a novel normal phase chromatographic condition. We improved upon our earlier assay by drastically reducing the instrument run time and eliminating the use of an ion pairing reagent. Stable isotope labeled substrates were utilized in the GALT and GALK assays. The enzymatic products ([ 13C 6]-uridine diphosphate galactose in GALT assay and [ 13C 6]-galactose-1-phosphate in GALK assay) were quantified in a 3 min LC–MS/MS run. The assays were sensitive enough to allow for the quantification of enzyme activities as low as 0.2% and 0.3% of normal control values in the GALT and GALK assays, respectively. Thirty-three samples from non-galactosemic patients were assayed to have erythrocyte GALT activity of 23.4 ± 4.2 and GALK activity of 1.8 ± 0.47 (mean ± SD) μmol⋅(g Hgb) − 1 h − 1 . Erythrocyte GALT activities in a cohort of 16 patients with classic or severe galactosemia were measured: 4 patients had GALT activity less than 1% of normal control values and the remaining 12 had no detectable GALT activity. No GALK activity was detected in a GALK deficient sample we analzyed. Lastly, we tested the feasibility of adapting this LC–MS/MS based GALT/GALK assay as a newborn screening (NBS) test.

The role of the thalamus in neuro-cognitive dysfunction in early unilateral hemispheric injury: A multimodality imaging study of children with Sturge–Weber syndrome

Background Sturge–Weber syndrome (SWS) with unilateral hemispheric involvement is a clinical model of early onset, chronic, often progressive hemispheric injury, resulting in variable neuro-cognitive impairment. Aims To evaluate if abnormal diffusion and metabolism of the thalamus, a central relay station with extensive cortical connections, may serve as a simple imaging marker of neuro-cognitive dysfunction in SWS. Methods We obtained both diffusion tensor imaging and FDG PET in 20 children (11 girls; age range: 3–12.4 years) with unilateral SWS. Diffusion parameters as well as FDG uptake were measured in thalami, compared to normal control values, and correlated with the extent of cortical hypometabolism, deep venous abnormalities and cognitive (IQ) as well as fine motor functions. Results Children with SWS had significantly higher thalamic glucose metabolic asymmetry than controls ( p = 0.001). Thalamic metabolic asymmetries correlated positively with the asymmetry of thalamic diffusivity ( p = 0.001) and also with the extent of cortical hypometabolism ( p < 0.001). Severe thalamic asymmetries of glucose metabolism and diffusion were strong predictors of low IQ (metabolism: p = 0.002; diffusivity: p = 0.01), even after controlling for age and extent of cortical glucose hypometabolism in children with left hemispheric involvement. Ipsilateral thalamic glucose hypometabolism was also associated with impairment of fine motor functions ( p = 0.002). Conclusions Both diffusion and glucose metabolic abnormalities of the thalamus are closely related to cognitive functions, independent of age and cortical metabolic abnormalities, in children with unilateral SWS. Thalamic metabolic asymmetry is a robust but simple imaging marker of neuro-cognitive outcome in children with early unilateral hemispheric injury caused by Sturge–Weber syndrome.

Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for GALT enzyme activity measurement. Our assay used stable isotope-labeled alpha- galactose-1-phosphate ([(13)C(6)]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([(13)C(6)]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [(13)C(6)]-Glu-1-P (265 > 79) as an internal standard. The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) mumol x (g Hgb)(-1) x h(-1) in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 micromol x (g Hgb)(-1) x h(-1) (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent K(m) of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. This LC-MS/MS-based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.

Determination of pipecolic acid following trimethylsilyl and trifluoroacyl derivatisation on plasma filter paper by stable isotope GC-MS for peroxisomal disorders

If early diagnosis is not made, patients with peroxisomal disorders rapidly progress to sudden death, physical defect or mental retardation resulted in storage of the toxic material into the brain. Therefore, it is necessary to develop the analytical method for rapid screening and/or correct confirmation diagnosis. The method utilizes [2H(9)]pipecolic acid as internal standard. The formation of trimethylsilyl derivative (TMS) of the carboxylic functional group was performed by adding MSTFA. And then 5 microL of methyl orange was added until the color of methyl orange was to yellow. Consecutively, the trifluoroacyl (TFA)-derivative of the -NH functional group was produced by adding MBTFA. GC-MS was set with specific ions (m/z 282, m/z 297) of the TMSTFA derivative of pipecolic acid for selected ion monitoring. The linearity of pipecolic acid in pooled plasma spots showed 0.9999 in the range of 10-150 ng investigated. The precision and accuracy was within S.D. of 5% (RSD, within 5%) for intra-day and inter-day assay. Normal control value has been determined in plasma spots of infant and adults aged 0-30 (including newborn). The utility of the method was demonstrated by quantifying various concentration of fortified pipecolic acid on a filter plasma spot. The new method was simple with just two step derivatisation, time and labor saving without SPE or liquid-liquid extraction, and convenience of delivery owing to the use of filter paper. The described method could be used for routine analysis, monitoring, and clinical diagnostic application of peroxisomal disorders on dietary therapy.

Immunodeficiency, Gastrointestinal Candidiasis, Wheat and Dairy Sensitivity, Abnormal Urine Arabinose, and Autism: A Case Study

A child with autism was found to have complete IgA deficiency (serum IgA <6 mg/dL; normal 33-235 mg/dL), Candidiasis of the gastrointestinal tract based on evaluation of stool testing and elevated urine arabinose, and elevated serum antibodies to wheat and dairy products. The pretreatment urinary arabinose concentration (341 mmol/mol creatinine in this child was nearly six times the mean value (60.4 mmol/mol creatinine, n=20) of normal children and over ten times the median value (31.0 mmol/mol creatinine) of normal controls. After antifungal therapy for four months, the urine was retested. At that time the urine arabinose was measured at 51 mmol/mol creatinine, a value only 15% of the baseline sample. Restriction of wheat and dairy products from the diet and antifungal therapy led to a significant decrease in autistic behaviors and increased rate of learning. The Childhood Autism Rating Scale (CARS), an observational measure of various aspects of autism, for the child has decreased from a rating of 43 (severely autistic) prior to introduction of these therapies to a value of 29 (non-autistic) after therapy.

ENU Mutagenesis Screening for Dominant Behavioral Mutations Based on Normal Control Data Obtained in Home-Cage Activity, Open-Field, and Passive Avoidance Tests

To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.

Phosphaturia as a Promising Predictor of Recurrent Stone Formation in Patients with Urolithiasis

PurposeRecent studies have suggested that renal phosphate leakage and the associated phosphaturia are significant underlying causes of calcium urolithiasis. The aims of this study were to assess whether phosphaturia relates to urinary metabolic abnormalities and recurrent stone formation.Materials and MethodsA database of patient histories and urine chemistries was analyzed for 1,068 consecutive stone formers (SFs) and 106 normal controls. Urine values for phosphaturia that were higher than 95% of the normal control values were defined as indicating hyperphosphaturia, and the effect of phosphaturia on urinary metabolites and stone recurrence was determined. Of these patients, 247 patients (23.1%) who had been followed up for more than 36 months or had a recurrence of stones during follow-up (median, 46.0 months; range, 5-151) were included in the analyses for stone recurrence.ResultsOf the SFs, 19.9% (212/1,068) had increased urinary phosphate excretion. SFs with hyperphosphaturia had a greater urinary volume and higher levels of calcium, uric acid, oxalate, and citrate than did SFs with normophosphaturia. A multivariate Cox regression model, stratified by stone episodes, revealed that hyperphosphaturia was an independent predictor of recurrent stone formation in first-time SFs (hazard ratio [HR]: 2.122; 95% confidence interval [CI]: 1.100-4.097; p=0.025). No association was detected between hyperphosphaturia and recurrent stone formation in recurrent SFs. Kaplan-Meier curves showed identical results.ConclusionsThis study demonstrates that hyperphosphaturia is closely associated with urinary metabolic abnormalities. Furthermore, hyperphosphaturia is a significant risk factor for stone recurrence in first-time SFs.