Abstract：
BACKGROUND:The determination of cellular beta-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for beta-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of beta-galactocerebrosidase activity can be performed following complete inhibition of beta-galactosidase activity. METHODS:We performed the assay using 2-7.5 microg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-beta-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO(3). Reactions were incubated for 30 min at 37 degrees C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (lambda(ex) 360 nm, lambda(em) 446 nm). RESULTS:AgNO(3) was a competitive inhibitor of beta-galactosidase [inhibition constant (K(i)) = 0.12 micromol/L] and completely inhibited beta-galactosidase activity when used at a concentration of 11 micromol/L. Under this condition, the beta-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect beta-galactocerebrosidase activity in as little as 2 microg cell protein extract or 7.5 microg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC(-/-) hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors. CONCLUSIONS:The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications.
journal_name
Clin Chemjournal_title
Clinical chemistryauthors
Martino S,Tiribuzi R,Tortori A,Conti D,Visigalli I,Lattanzi A,Biffi A,Gritti A,Orlacchio Adoi
10.1373/clinchem.2008.115873subject
Has Abstractpub_date
2009-03-01 00:00:00pages
541-8issue
3eissn
0009-9147issn
1530-8561pii
clinchem.2008.115873journal_volume
55pub_type
杂志文章abstract：:The concentration of the conjugated bile acid, cholylglycine, in serum is a sensitive and specific indicator of hepatic function. We describe a convenient, specific, and precise radioimmunoassay for cholylglycine, in which 125I-labeled cholylglycyltyrosine is used as tracer. In addition, a blocking agent in the buffer...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1981-10-01 00:00:00
abstract：:Two patients with Paget's disease of bone were subjected to plasmapheresis. Alkaline phosphatase activities of serum declined sharply, but returned to preplasmapheresis values within eight to 10 days. The biological half-life of circulating skeletal alkaline phosphatase, as calculated from these experiments, is betwee...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1982-01-01 00:00:00
abstract：:A new method for the reliable identification of localized variations in DNA by detection of associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The diagnostic products are generated by the primer oligo base extension (PROBE) reaction, which req...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1997-07-01 00:00:00
abstract：BACKGROUND:Technological advances have resulted in a renaissance of proteomic studies directed at finding markers of disease progression, diagnosis, or responsiveness to therapy. Renal diseases are ideally suited for such research, given that urine is an easily accessible biofluid and its protein content is derived mai...
journal_title：Clinical chemistry
pub_type： 杂志文章,评审
doi：10.1373/clinchem.2011.165969
更新日期：2012-02-01 00:00:00
abstract：:We studied the effects on 25 analytes of duration of contact of serum with non-anticoagulated blood and of temperature. Serum was separated after blood was allowed to stand, for 0, 2, 4, 6, 8, 24, or 48 h at 4, 23, or 30 degrees C. Results obtained for bilirubin, albumin, zinc sulfate turbidity, thymol turbidity, chol...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1981-01-01 00:00:00
abstract：:We report a method for the extraction of angiotensin peptides from plasma with a mixture of acetone, 1 mol/L HCl, and water (40/1/5 by vol). The method is highly reproducible for the measurement of angiotensin I and angiotensin II in small sample volumes, with analytical recoveries of about 80% for both peptides. We i...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1988-06-01 00:00:00
abstract：:We have used a monoclonal antibody-based ELISA for plasma prothrombin fragment 1.2 (F1.2) to establish appropriate sample collection and storage conditions for this biomarker of thrombin generation. F1.2 concentrations were not altered by exogenous factor Xa, thrombin, or thromboplastin if blood was collected by routi...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1994-10-01 00:00:00
abstract：:A simple computer-based screening technique has been developed for classifying human expired air components into 16 chemical classes, based on empirical formulas. The sort procedure was developed to simplify the screening of the composition of expired air samples by sorting all components into chemical classes and cla...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1988-08-01 00:00:00
abstract：:We compared a stabilized-temperature L'vov platform furnace containing an end-heated graphite atomizer (HGA) and transverse Zeeman background-correction system with a side-heated furnace system (transversely heated graphite atomizer; THGA) containing a longitudinal Zeeman background-correction system for the determina...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1994-03-01 00:00:00
abstract：:We assessed several methods of inorganic phosphate assay for their suitability in estimating phospholipids in digested extracts of amniotic fluids. The extraction and digestion procedures used for phospholipids from amniotic fluid were also examined critically. The effect of contamination by blood or obstetric cream h...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1975-09-01 00:00:00
abstract：:We describe a new and specific method for measurement of lecithin palmitic acid in amniotic fluid. Dipalmitoyl lecithin, the major alveolar surfactant, has previously been estimated by measuring the lecithin-sphingomyelin ratio, total lecithin, total phospholipid phosphorus, and (or) total palmitic acid. Our method is...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1977-01-01 00:00:00
abstract：:In this new method for determining urinary protein, the reaction is complete within 10 min at 37 degrees C. This method is applicable to automated as well as manual measurements. Protein concentration and absorbance at 600 nm are linearly related throughout a wide range of concentrations, 10 to 16 000 mg/L. However, t...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1986-08-01 00:00:00
abstract：BACKGROUND:Increased plasma troponin T (cTnT), but not troponin I (cTnI), is frequently observed in end-stage renal failure patients. Although generally considered spurious, we previously reported an associated increased mortality at 12 months. METHODS:We studied long-term outcomes in 244 patients on chronic hemodialy...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：2001-03-01 00:00:00
abstract：:Single-strand conformation polymorphism (SSCP) analysis is widely used to prescreen mutations in p53 gene. However, standardization of SSCP to detect p53 mutations has rarely been pursued so far. We have developed complete conditions for a temperature-controlled nonradioactive SSCP for mutation detection in amplified ...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1997-12-01 00:00:00
abstract：BACKGROUND:Little research has been done to examine whether gamma-glutamyltransferase (GGT) is prospectively associated with the development of chronic kidney disease (CKD). We performed a prospective study to examine the association between GGT and the risk for the development of CKD. METHODS:The study cohort include...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2006.078980
更新日期：2007-01-01 00:00:00
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journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1987-08-01 00:00:00
abstract：:The isoenzyme pattern of N-acetyl-beta-glucosaminidase (NAG) in serum and urine was studied in two groups of patients with diabetes mellitus and in 30 control subjects. Total NAG activity was significantly (P less than 0.001) increased in the serum and urine of the 20 diabetics with vascular complications, but was ins...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1988-12-01 00:00:00
abstract：BACKGROUND:Several plasma proteins have been suggested as markers for a variety of cardiovascular conditions but fail to qualify in independent patient cohorts. This may relate to interference of medication on plasma protein concentrations. We used proteomics to identify plasma proteins that changed in concentration wi...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2017.282665
更新日期：2018-10-01 00:00:00
abstract：BACKGROUND:This Case Conference reviews the normal changes in thyroid activity that occur during pregnancy and the proper use of laboratory tests for the diagnosis of thyroid dysfunction in the pregnant patient. CASE:A woman in the 18th week of pregnancy presented with tachycardia, increased blood pressure, severe vom...
journal_title：Clinical chemistry
pub_type： 杂志文章,评审
doi：
更新日期：1999-12-01 00:00:00
abstract：BACKGROUND:C-reactive protein (CRP), the prototypic marker of inflammation, is present in atherosclerotic plaques and appears to promote atherogenesis. Also, CRP has been localized to monocytes and tissue macrophages, which are present in the necrotic core of lesions prone to plaque rupture. Leukocyte-derived myelopero...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：10.1373/clinchem.2008.109207
更新日期：2009-02-01 00:00:00
abstract：:Radioimmunoassay of triiodothyronine was performed with a homogeneous incubation system. Bound and free ligand were separated by partitioning in an aqueous two-phase system. The agreement between the results obtained with this technique and conventional heterogeneous radioimmunoassay was very good (r = 0.981). ...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1982-04-01 00:00:00
abstract：:Measurement of colloid osmotic pressure complements measurements of pulmonary artery wedge pressure for assessing the risks of pulmonary edema and constitutes an increasingly important reference for purposes of guiding selection of colloid or crystalloid fluids in patients with acute cardiac disease. We describe a sim...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1978-09-01 00:00:00
abstract：:3-Hydroxy-3-methylglutaric aciduria was detected in a newborn. The progress of the dietary therapy for the disorder was monitored by dual-column "high-performance" liquid chromatography with a computer-controlled photodiode array spectrophotometric detector. This procedure is a quick way to detect and monitor the prog...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1986-01-01 00:00:00
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journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1991-12-01 00:00:00
abstract：:A literature review of the effects of long-term freezer storage on concentrations of antioxidant micronutrients in serum or plasma showed that a high proportion of the studies that compared the concentrations of retinol, beta-carotene, or alpha-tocopherol in fresh and stored serum were deficient because of small numbe...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1993-06-01 00:00:00
abstract：:We describe specifications for high-purity 4-nitrophenol, which is suitable for spectrophotometric standardization. Such a reference material is needed in clinical enzymology to establish the proper molar absorptivity of 4-nitrophenol under final reaction conditions, particularly for measuring alkaline phosphatase act...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1980-05-01 00:00:00
abstract：:We examined the mechanism by which hypertriglyceridemia interferes with the Roche Diagnostics "Amylochrome" procedure for measurement of amylase in lactescent samples with normal and above-normal amylase activity. The serum blank recommended in the Amylochrome protocol to compensate for lactescence remains inappropria...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1982-10-01 00:00:00
abstract：:We used ion-exchange column chromatography and electrophoresis on polyacrylamide gel to compare the acid phosphatase isoenzymes of prostate and leukocytes. The major isoenzyme of the prostate is band 2A; only a trace of band 2B was observed. However, the major isoenzymes of leukocytes are band 4 and band 2B, and only ...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1979-07-01 00:00:00
abstract：:Two-dimensional electroimmunodiffusion is rarely used in clinical chemistry, because its potentialities are not yet widely appreciated. Studying mouse anaphylaxis, we observed with this technique certain striking short- and long-term pathologic changes in several important serum antigens, which aptly demonstrate its u...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1979-05-01 00:00:00
abstract：:We describe a rapid method for determining cholesterol sulfate in plasma and erythrocytes. After its single-step isolation by means of anion-exchange chromatography cholesterol sulfate is hydrolyzed, trimethylsilylated, and determined by gas chromatography with flame ionization detection. 5 beta-Cholestan-3 alpha-ol s...
journal_title：Clinical chemistry
pub_type： 杂志文章
doi：
更新日期：1983-07-01 00:00:00
