Specific Substrate for the Assay of Lysosomal Acid Lipase.
LAL is detected in leukocytes or fibroblasts using radiometric, immunologic, or fluorescence assays (4). Hamilton and colleagues recently developed a fluorometric assay to detect LAL in dried blood spots (DBS) (5). The assay is based on the release of 4-methylumbelliferone from its ester with palmitic acid. However, the substrate is not specific for LAL, and the method involves 2 assays performed in parallel, 1 in the presence of an LAL-specific covalent inactivator Lalistat-2 (5). LAL activity is calculated from the difference in these 2 assays.
Recently, enzyme replacement therapy with recombinant LAL (sebelipase a) has been approved by the Food and Drug Administration for management of LAL deficiency (6). As with many lysosomal storage diseases, initiation of treatment before the onset of irreversible symptoms may be advantageous. Thus, newborn screening for LAL deficiency may be warranted. However, given the rarity of LAL deficiency, newborn screening for this enzyme might be practical if it could be added in a multiplex fashion to an existing lysosomal storage disease newborn screening panel, if it could be done inexpensively, and if the number of follow-up samples was minimal. Here we describe the discovery of a substrate that is selective for LAL in DBS, thereby allowing the activity of this enzyme to be measured in a single incubation.
Materials and Methods
MATERIALS AND METHODS
All patient samples were obtained with Institutional Review Board approval from the University of Washington. DBS from anonymized samples from LAL-deficient patients were obtained from Dr. Rhona Jack (Seattle Children's Hospital). All patient samples were stored at 4 [degrees]C in sealed plastic bags. Other DBS samples were stored in plastic bags at -20 [degrees]C in sealed containers with desiccant. Recombinant LAL was obtained as a gift from Alexion Corp. The synthesis of all new reagents is detailed in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol64/ issue4. Lalistat-2 was provided as a gift from Dr. J. Hamilton (Yorkhill Hospital, UK) or synthesized as described (7).
LC-MS/MS LAL ASSAY
Assay mixture was prepared by combining 9 volumes of 0.1 mol/L sodium acetate buffer (made by adding reagentgrade NaOH to 0.1 mol/L reagent-grade acetic acid in water), pH 4.5, containing 2.5 mmol/L sodium taurodeoxycholate hydrate (Sigma, 287245) with 1 volume of 0.5% (w/v) bovine heart cardiolipin in ethanol (Sigma, C1649) containing 3 mmol/L substrate 4-propyl-8-methyl-7-hydroxycoumarin (P-PMHC) and 50 [micro]mol/L carbon-13-labeled 4-propyl-8-methyl-7hydroxycoumarin (internal standard). The latter was prepared by mixing solid substrate P-PMHC with internal standard solution in absolute ethanol, removing solvent in a centrifugal concentrator under vacuum (or with a jet of nitrogen), and then mixing with commercial cardiolipin/ethanol solution. This ethanol stock could be stored at -20 [degrees]C for several months (avoid >2--3 freeze-thaw cycles), and the aqueous buffer could be stored at 4 [degrees]C for several months. This was based on no change in assay results when new and stored solutions were used (not shown). Assay mixture was made fresh before each use.
To a 3-mm punch from a DBS in a 1.5-mL polypropylene Eppendorf tube was added 200 [micro]L of purified water (Milli-Q, EMD Millipore). The tubes were shaken on an orbital platform shaker for 1 h at ambient temperature. The tube of DBS extract was mixed briefly on a vortex-type mixer, and a 10-[micro]L aliquot was added to the well of a polypropylene, deep-well, 96-well plate (Costar, 3959). Assay mixture (30 [micro]L, ambient temperature) was added to each well. The plate was centrifuged at 3000g for 5 min at ambient temperature to ensure that all liquid was at the well bottom. The plate was sealed with a film cover (Advantage 96-well round clear Silicone/PTFE cap mat 962611). The plate was shaken at 37 [degrees]C in an incubator with orbital mixing at 400 rpm for 3 h. The reactions were quenched by addition of 80 [micro]L of purified water (Milli-Q, EMD Millipore) followed by 400 [micro]L of HPLC-grade ethyl acetate. The well contents were mixed by pipetting up and down approximately 10 times. The plate was centrifuged at 3000g for 5 min at ambient temperature, and then 120 [micro]L of the upper ethyl acetate layer was transferred to a shallow-well, 96-well polypropylene plate (Greigner, 651201). Solvent was removed at ambient temperature with a jet of [N.sub.2]. To each well was added 200 [micro]L of water/methanol (1:1, Fisher Optima Grade), and samples were mixed by pipetting up and down a few times. The plate was wrapped with aluminum foil and placed in the autosampler chamber at 8 [degrees]C in preparation for LC-MS/MS analysis.
Liquid chromatography was carried out using a Waters Acquity binary solvent pump system with a CSH, C18, 1.7-[micro]m, 2.1 X 50-mm column (Waters, 186005296) and a CSH, C18, 1.7-[micro]m guard column (Vanguard, 186005303). The solvent program was 99% A (70:30, water/acetonitrile, 0.1% formic acid, Fisher Optima grade)/1% B (50:50, acetonitrile/ isopropanol, 0.1% formic acid) to 30% A/70% B over 1 min, then jump to 100% B and hold for 0.5 min, then to 99% A over 0.5 min (flow rate 0.8 mL/min). The total run time was 2.5 min. Mass spectrometry was carried out with a Waters Xevo TQ tandem-quadrupole instrument. Additional liquid chromatography and tandem mass spectrometry parameters are provided in Table 1 of the online Data Supplement.
FLUOROMETRIC LAL ASSAY
The fluorometric assay up to the end of the incubation was identical to the LC-MS/MS assay, except no internal standard was present in the stock substrate solution. After incubation, the fluorometric assay was continued as follows: Reactions were quenched by addition of 200 [micro]L of water/methanol (1:1, Fisher Optima Grade). The well contents were mixed by pipetting up and down approximately 10 times. A portion (150 [micro]L) of the well contents was transferred to a black, flat-bottomed 96-well microtiter plate (NUNC 437112). Samples were immediately read on a fluorometer (PerkinElmer Victor3V 1420) with an excitation wavelength of 355 nm, an emission wavelength of 460 nm, and an excitation time of 0.1 s. The fluorometer reading was converted to micromoles of product by generating a standard curve. DBS extract (60 [micro]L) and assay mixture (450 [micro]L) were incubated separately. To a well was added 200 [micro]L of water/methanol solution (1:1), 10 [micro]L of DBS extract, and 30 [micro]L of assay mixture. To each well was added 2 [micro]L of LAL product (4-propyl-8-methyl-7-hydroxycoumarin) (0, 0.2, 1, or 2 nmol) from an aqueous stock solution. After mixing by pipetting up and down approximately 10 times, a 150-pL aliquot was transferred to the fluorometer plate and submitted to fluorometry as above. The well in which no product 2 was added serves as the blank. Note that because DBS extract and assay mixture were incubated separately, this blank reflected (a) the fluorescence of the blood extract and substrate; (b) any fluorescence owing to nonenzymatic breakdown of substrate; and (c) any quenching of the fluorescence by components of the blood.
DEVELOPMENT OF A NEW LAL ASSAY
Our goal was to find a new substrate that was acted upon only by LAL in DBS, thus avoiding the need to carry out 2 assays in parallel in the presence and absence of the LAL-specific inactivator Lalistat-2 (5). The most obvious choice was to consider the natural LAL substrates, cholesterol esters, and triglycerides. By carrying out the published assay (5) with palmitoyl-4MU on a DBS extract and also with known amounts of recombinant LAL, we determined that there was no more than 50 to 100 pg of LAL in the DBS extract. Using this number and the published specific activities for purified LAL acting on the natural substrates cholesterol oleate and triolein (8), we concluded that the amount of products produced with a 3-mm DB punch and the natural substrates would be well below the detection limits for mass spectrometry. Thus, assays using these natural substrates were not attempted.
The next stage was to test a variety of new fatty acid esters as possible substrates for LAL (see the online Data Supplement). We first tested variants of palmitoyl-4MU containing replacements for the 4MU group that were structurally similar to the leaving groups we used in our previously designed substrates for other lysosomal enzymes (9) (1, see online Data Supplement). We also tested the ester formed between cholesterol and a fatty acyl group containing a bis-amide at the w end of the acyl chain (2, see online Data Supplement). Next, we tested analogs of palmitoyl-4MU containing alkyl-amide chains attached to the methyl of the 4MU moiety (10) (3, 5 see online Data Supplement). Finally, we tested analogs of palmitoyl-4MU containing bis-amides at the w terminus of the fatty acyl group of palmitoyl-4MU (4, 6, see online Data Supplement). None of these compounds displayed detectable LAL activity when tested with DBS under the conditions given in the online Data Supplement. These studies show that addition of polar groups to the palmitoyl chain abrogated activity toward LAL. Also, extension of the 4MU moiety with polar chains was also not tolerated.
At this stage, the only substrate that displayed measurable LAL activity using DBS was palmitoyl-4MU. This is presumably because of the highly reactive nature of the ester linkage to the strong leaving group 4MU and the fact that LAL prefers highly hydrophobic substrates. Thus, we turned to the preparation of a library of compounds that contained relatively small structural variations of the palmitoyl-4MU substrate. The structures of all library components are shown in Table 3 of the online Data Supplement. From this library of 25 compounds, P-PMHC (Fig. 1) emerged as the substrate with the best combination of high specific activity on LAL and high specificity for LAL. Thus, we carried out additional studies with P-PMHC.
We studied a variety of phospholipids as replacements for cardiolipin, which was used in the original LAL assay (5). The following phospholipids were tested individually at 0.1 mmol/L: 1,2-dioleoyl-phosphatidylethanolamine, 1,2-dioleoyl-phosphatidylmethanol, 1,2-dioleoyl-phosphatidylcholine, 1,2-dioleoyl-phosphatidylserine, brain phosphatidylethanolamine, 1,2-heptadecyl-phosphatidylcholine, and 1,2-lauryl-phosphatidylcholine. None of these phospholipids led to any substantial increase in LAL over that measured with cardiolipin (data not shown); thus, we continued our studies with the latter.
In the original assay (5), DMSO was used as a solvent to prepare the palmitoyl-4MU and Lalistat-2 stock solutions. The final concentration of DMSO in the LAL assay was 2.4%. Over the course of our studies, we noticed that the quality of the DMSO was critical to being able to detect LAL activity in DBS. Previously opened bottles of DMSO stored for approximately 1 month or longer led to essentially complete loss of LAL activity measured in DBS. Based on this result, we decided to replace DMSO initially with dimethylformamide for preparation of the substrate stock solution, and to use ethanol to prepare the Lalistat-2 solution (however, with the use of LAL-specific P-PMHC, Lalistat-2 was no longer used). In our final assay conditions, we used ethanol as the only solvent other than water.
The previous LAL assay used Triton X-100 as the detergent (5). We replaced this with sodium taurodeoxycholate because this detergent largely remains in the aqueous phase following liquid-liquid extraction with ethyl acetate (thus minimizing injection of large amounts of detergent onto the UPLC column). Replacement of Triton X-100 with 2.5 mmol/L sodium taurodeoxycholate resulted in a 20% decrease of LAL activity, and increasing the detergent concentration further led to an additional loss of activity. Thus, 2.5 mmol/L was chosen for all subsequent studies.
The LAL pH rate profile was studied from 3.5 to 7.0 in 0.1 mol/L succinate or from 3.5 to 5.5 in 0.1 mol/L sodium acetate (Fig. 2). Based on this study, we chose 0.1 mol/L sodium acetate, pH 4.5 to maximize LAL activity.
We settled on a concentration of P-PMHC in the mixture of 3 mmol/L based on a good combination of high LAL activity and minimal background (no DBS control, datanot shown). The Hamilton assay (5) used 40 [micro]L of an aqueous extract of a 3-mm DBS added to 160 [micro]L of assay mixture. To keep the solvent volumes for the liquid-liquid extraction to a minimum, thus avoiding long times for solvent removal, we added 10 [micro]L of an aqueous extract of a 3-mm DBS punch to 30 [micro]L of assay mixture.
With the final assay conditions chosen, we obtained a specific activity of 874 [micro]mol/h/L using a 3-mm punch of a DBS from a healthy adult. The activity was measured by UPLC-MS/MS after extraction of the reaction mixture with ethyl acetate. The tandem mass spectrometry multiple reaction monitoring response was converted to micromoles of product with the use of a chemically identical but isotopically differentiated internal standard (carbon 13-labeled 4-propyl-8-methyl-7-hydrdoxycoumarin) (Fig. 1). The use of the internal standard accounts for all product losses owing to sample processing and analysis. Using a 3-mm punch from an identical DBS and the previous fluorometric LAL assay with and without Lalistat-2 (5), we obtained a specific activity of 466 [micro]mol/h/L (within the range of values reported previously) for the LAL component, which is 1.9-fold lower than the activity measured with the new UPLC-MS/MS assay.
The following studies established that LAL substrate P-PMHC was highly specific for LAL in DBS (Table 1). When extracts from 2 healthy persons' DBS were prein cubated with 10 [micro]mol/L Lalistat-2, 98% of the activity toward P-PMHC was inhibited. This increases to 99% inhibition if 100 [micro]mol/L Lalistat-2 was used (Table 1). In contrast, when the same DBS extract was submitted to the previously reported assay with palmitoyl-4MU (5), 78% of the total lipase activity was blocked by 10 [micro]mol/L Lalistat-2 (not shown). Under the assumption that Lalistat-2 was completely selective for LAL, the data showed that P-PMHC, but not palmitoyl-4MU, was highly specific for LAL in DBS.
FLUOROMETRIC LAL ASSAY
Because hydrolysis of P-PMHC leads to the fluorescent product 4-propyl-8-methyl-7-hydroxycoumarin, we used a standard plate reader fluorometer to measure LAL activity in DBS. Table 2 shows LAL activities measured with UPLC-MS/MS and by fluorometry on an identical set of DBS from 10 adults. Agreement between the 2 assays generally showed <30% difference in activity values for all but 1 pair, for which the difference approached 50%.
The analytical range is an important assay parameter that is defined as the ratio of assay response measured with the quality control high sample (typical of a healthy person) divided by the assay response for all elements independent of LAL (11). The larger the analytical range, the greater the activity values will be spread out, leading to greater accuracy especially when activity is low. The mean analytical range for the UPLC-MS/MS assay was 44 compared with a value of 14 for the fluorometric assay.
STUDIES WITH PATIENT SAMPLES
Fig. 3 shows the LAL activity data in DBS from 15 healthy children (<5 years old), 12 healthy adults, and 6 patients previously shown to be LAL-deficient. All patients had symptoms consistent with LAL deficiency, and these 6 patients were shown to be LAL-deficient by the original LAL assay method on DBS (5). The patients are deidentified, and we have no additional information.
All assays were carried out with UPLC-MS/MS using the optimized conditions with substrate P-PMHC in the absence of Lalistat-2 (thus, only a single assay per patient was needed). Individual values are shown in Table 4 of the online Data Supplement. All healthy persons showed LAL activity well separated from that measured with LAL-deficient patients. The fact that LAL activity was close to zero in all 6 LAL-deficient patients was further evidence that substrate P-PMHC was LAL-specific.
The new LAL assay makes use of a substrate that is highly specific for LAL, thus allowing its activity to be measured in DBS using a single incubation. In contrast, about onethird of the standard substrate used to assay LAL, palmitoyl-4MU, is hydrolyzed by [greater than or equal to]1 esterases/lipases in DBS other than LAL. In the original assay, the LAL activity is obtained as the fraction of palmitoyl-4MU hydrolase activity that is sensitive to Lalistat-2 (5). Because the LAL activity component is obtained as the difference in 2 substantial activity values, the error in LAL activity must include the propagation of errors in 2 separate measurements. Thus, it would be required to raise the cutoff value in a newborn screening program based on the Lalistat-2 method. This would result in an increase in the false-positive rate. With the new LAL-specific substrate P-PMHC, not only can the effort to perform the assay be reduced, but the screen cutoff can also be lowered.
It has been shown by comparison of large pilot newborn screening studies of lysosomal storage diseases that the tandem mass spectrometry enzymatic activity method gives a substantially lower number of positive screenings than the fluorometric method when compared at equivalent cutoff values (12). This may be because the analytical range of the tandem mass spectrometry assays is > 3-fold greater than that of the corresponding fluorometric assays (11). Thus, the use of UPLC-MS/MS may be the method of choice for newborn screening of LAL deficiency, but this remains to be determined. Finally, it should be possible to multiplex the new UPLC-MS/MS LAL assay with other tandem mass spectrometry assays for lysosomal storage diseases simply by addition of substrate P-PMHC to a single assay mixture containing a collection of additional substrates and internal standards and performing a single UPLC-MS/MS in which all products and internal standards are detected by multiple reaction monitoring mode.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: M.H. Gelb, University of Washington. Consultant or Advisory Role: C.R. Scott, Genzyme Corp; M.H. Gelb, PerkinElmer.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: S. Masi, National Institutes of Health (F30 DH081853), National Institutes of Health (R01 DK067859),N.K. Chennamaneni, National Institutes of Health (R01 DK067859); F. Turecek, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health (R01 DK067859); C.R. Scott, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health (R01 DK067859); M.H. Gelb, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health (R01 DK067859). Seattle Children's Hospital provided DBS from LAL-deficient patients; Yorkhill Hospital, UK, provided Lalistat-2; Alexion Corp. provided recombinant LAL. Expert Testimony: None declared.
Patents: None declared.
Role of Sponsor: The funding organizations played a direct role in the design of study, choice of enrolled patients, review and interpretation of data, and final approval of manuscript.
Acknowledgments: The authors thank Dr. Rhona Jack (Seattle Children's Hospital) for DBS from LAL-deficient patients, J. Hamilton (Yorkhill Hospital, UK) for Lalistat-2, andAlexion Corp. for recombinant LAL.
(1.) Aguisanda F, Thorne N, Zheng W. Targeting Wolman disease and cholesterol ester storage disease: disease pathogenesis and therapeutic development. Curr Chem GenomTransl Med 2017;11:1-18.
(2.) Canaan S, Roussel A, Verger R, Cambillau C. Gastric lipase: crystal structure and activity. Biochim Biophys Acta 1999;1441:197-204.
(3.) Rajamohan F, ReyesAR, Ruangsiriluk W, Hoth LR, Han S, Caspers N, et al. Expression and functional characterization of human lysosomal acid lipase gene (LIPA) mutation responsible for cholesteryl ester storage disease (CESD) phenotype. Prot Expr Purif 2015;110:22-9.
(4.) Gravel RA, Kaback MM, Proia RL, Sandhoff K, Suzuki K. The metabolic & molecular bases of inherited disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler B, et al., editors. The metabolic and molecular bases of inherited disease. 8th Ed. New York (NY): McGraw-Hill; 2001. p. 3827-3387.
(5.) Hamilton J, Jones I, Srivastava R, Galloway P. A new method for the measurement of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2. Clin Chim Acta 2012;413:1207-10.
(6.) Jones SA, Rojas-Caro S, Quinn AG, Friedman M, Marulkar S, Ezgu F, et al. Survival in infants treated with sebelipase alfa for lysosomal acid lipase deficiency: an open-label, multicenter, dose-escalation study. Orphanet J Rare Disl 2017;12:25.
(7.) Ebdrup S, Refsgaard HHF, Fledelius C, Jacobsen P. Synthesis and structure-activity relationship for a novel class of potent and selective carbamate-based inhibitors of hormone selective lipase with acute in vivo antilipolyticeffects. J Med Chem 2007;50:5449-56.
(8.) Negre A, Salvayre R, Dagan A, Borrone C, Gatt S. New spectrophotometric assays of acid lipase and their use in the diagnosis of Wolman and cholesteryl ester storage diseases. Anal Biochem 1985;145:398-405.
(9.) Li Y, Scott CR, Chamoles NA, Ghavami A, Pinto BM, Turecek F, et al. Direct multiplex assay of lysosomal en zymes in dried blood spots for newborn screening. Clin Chem 2004;50:1785-96.
(10.) Wolfe BJ, Blanchard S, Sadilek M, Scott CR, Turecek F, Gelb MH. Tandem mass spectrometry for the direct assay of lysosomal enzymes in dried blood spots: application to screening newborns for mucopolysaccharidosis II (Hunter syndrome). Anal Chem 2011;83:1152-6.
(11.) Kumar AB, Spacil Z, Masi S, Ghomashchi F, Ito M, Scott CR, et al. Tandem mass spectrometry has a larger analytical range than fluorescence assays of lysosomal enzymes: application to newborn screening and diagnosis of mucopolysaccharidoses types II, IVA, and VI. Clin Chem 2015;61:1363-71.
(12.) Elliot S, Buroker N, Cournoyer J, Potier A, Trometer J, Elbin C, et al. Pilot study of newborn screening for six lysosomal storage diseases using tandem mass spectrometry. Mol Genet Metab 2016;118:304-9.
Sophia Masi,  Naveen Chennamaneni,  Frantisek Turecek,  C. Ronald Scott,  and Michael H. Gelb  *
 Department of Chemistry, University of Washington, Seattle, WA;  Department of Pediatrics, University of Washington, Seattle, WA.
* Address correspondence to this author at: Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195. Fax 206-685-8665; e-mail firstname.lastname@example.org. Received September 21,2017; accepted December 29,2017.
Previously published online at DOI: 10.1373/clinchem.2017.282251
 Nonstandard abbreviations: LAL, lysosomal acid lipase; DBS, dried blood spot on a newbornscreening card; P-PMHC, 4-propyl-8-methyl-7-hydroxycoumarin.
 Human gene: LIPA, lipase A.
Caption: Fig. 1. Structure of LAL-specific substrate P-PMHC, LAL product, and heavy atom substituted internal standard.
Caption: Fig. 2. pH rate profile for recombinant human LAL activity using substrate P-PMHC from pH 3.5 to 7.0: complete assay in 0.1 mol/L succinate (filled circles); complete assay in 0.1 mol/L sodium acetate (open circles); no enzyme blank in 0.1 mol/Lsuccinate (filled triangles); complete assay in 0.1 mol/L succinate with Lalistat-2 inhibition (open squares). Buffer (9 volumes) was combined with 2.5 mmol/L sodium taurodeoxycholate, 3.7 mmol/L substrate P-PMHC, and 1 volume of 0.5%(w/v) cardiolipin in ethanol. Full assays were carried out with DBS extract from a healthy adult and in the absence of Lalistat-2. Blank assays were carried out using water and 300 [micro]mol/L Lalistat-2. Inhibited assays were carried out as for the full assays but in the presence of 300 [micro]mol/L Lalistat-2.
Caption: Fig. 3. LAL activity in DBS measured with substrate P-PMHC and UPLC-MS/MS.
The bottom of each box is the value of the first quartile, the middle line is the medium value, and the top line is the third quartile. All values are blank corrected. Individual patient values are given in Table 4 of the online Data Supplement.
Table 1. Effect of Lalistat-2 on the activity (tandem mass spectrometry method) of LAL toward substrate P-PMHC. (a) DBS Activity without Activity with 10 Lalistat-2, [micro]mol/L [micro]mol/h/L Lalistat-2, [micro]mol/h/L Adult 1 (triplicate) 554, 582, 582 10, 9, 10 Adult 2 (triplicate) 519, 539,473 5, 2, 3 Blank (15 measurements) 16.8-26.2 DBS Activity with 100 [micro]mol/L Lalistat-2, [micro]mol/h/L Adult 1 (triplicate) 6, 4, 5 Adult 2 (triplicate) 2, 0, 1 Blank (15 measurements) (a) Activity values for the adult DBS were blank corrected. The blank was obtained by using an equal volume of water instead of water-extracted DBS. Table 2. LAL activity in DBS measured by UPLC-MS/MS and fluorometric assays. Type Samples LAL activity, [micro]mol/h/L blood (a) Fluorescence Adult 1 746, 707, 596, 599 2 541, 751, 675, 519, 803 3 618, 592, 583, 628 4 933, 1683, 1073, 938, 1261, 1348 5 809, 917, 901, 768, 815 6 1198, 1635, 1361, 1382, 1284 7 758, 854, 841, 645, 781 9 427, 100, 376, 239 10 767, 857, 1150, 1158, 900 12 989, 843, 792, 918, 604, 703 Blank (b) 48-78 UPLC-MS/MS Adults 1 598, 555, 653, 578, 608 2 712, 701, 674, 632, 588, 636 3 492, 499, 506, 480, 453, 465 4 962, 842, 862, 848, 1016, 849 5 846, 852, 852, 864, 923, 907 6 990,979, 1129, 1152,929 7 613, 555, 488, 536,514,470 9 316, 330, 220, 247, 292, 236 10 938, 796,818, 627,1112, 740 12 424, 534,376, 496, 368, 385 Blank (b) 6.3-24.9 Type Samples Mean %CV Fluorescence Adult 1 662 9.94 2 658 17.08 3 605 3.05 4 1206 21.81 5 842 6.83 6 1372 10.67 7 794 9.95 9 286 44.54 10 980 15.89 12 808 15.89 Blank (b) 58 19.03 UPLC-MS/MS Adults 1 598.5 5.5 2 657.1 6.5 3 482.2 3.9 4 896.3 7.5 5 874.0 3.4 6 1035.8 8.5 7 529.0 8.9 9 273.4 15.2 10 838.3 18.3 12 430.4 14.7 Blank (b) 15 36.4 (a) Activity values are blank corrected. (b) Range of blank values given for 12 and 56 repeats of the fluorometric and tandem mass spectrometry assays, respectively.
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|Title Annotation:||Automation and Analytical Techniques|
|Author:||Masi, Sophia; Chennamaneni, Naveen; Turecek, Frantisek; Scott, C. Ronald; Gelb, Michael H.|
|Date:||Apr 1, 2018|
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