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A review paper on quantification of albumin

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Quantification of Urinary Albumin by Using Protein;Cleavage and LC-MS/MS;Jesse C. Seegmiller,1;David R. Barnidge,1 Bradley E. Burns,1 Timothy S. Larson,1,2,3;John C. Lieske,1,2;and Rajiv Kumar2,3,4,5*;BACKGROUND;Urinary albumin excretion is a sensitive;diagnostic and prognostic marker for renal disease.;Therefore, measurement of urinary albumin must be;accurate and precise. We have developed a method to;quantify intact urinary albumin with a low limit of;quantification (LOQ).;METHODS;We constructed an external calibration;curve using purified human serum albumin (HSA);added to a charcoal-stripped urine matrix. We then;15;added;an;internal;standard;N-labeled;recombinant HSA (15NrHSA), to the;calibrators, controls, and pa- tient urine;samples. The samples were reduced, alkylated, and digested with trypsin. The;concentration of albumin in each sample was;determined by liquid chromatographytandem;mass spectrometry (LC-MS/ MS) and linear;regression analysis, in which the relative;abundance area ratio of the tryptic;peptides;42;LVNEVTEFAK51 and 526QTALVELVK534 from;albu- min and 15NrHSA were referenced to the;calibration curve.;RESULTS;The lower limit of quantification was 3.13;mg/L, and the linear dynamic range was 3.13200;mg/L. Replicate digests from low, medium, and high;controls (n = 5) gave intraassay imprecision CVs of;2.8%11.0% for the peptide 42LVNEVTEFAK51;and;QTALVELVK534 peptide.;In- terassay imprecision of the controls for a;period of 10 consecutive days (n = 10) yielded;CVs of 1.5%14.8% for the 42LVNEVTEFAK51;peptide, and 6.4%14.1% for;the;526;QTALVELVK534 peptide. For the;42;LVNEVTEFAK51;peptide;a;method;comparison be- tween LC-MS/MS and an;immunoturbidometric method for 138 patient;samples gave an R2 value of 0.97 and a;regression line of y = 0.99x + 23.16.;1.9%12.3% for the;CONCLUSIONS;526;Urinary albumin can be quantified by a;protein cleavage LC-MS/MS method using a 15NrHSA;internal standard. This method provides improved analytical performance in the clinically relevant range;compared to a commercially available immunoturbidometric assay.;2008 American Association for Clinical Chemistry;The accurate and precise measurement of urinary albumin excretion is important in clinical medicine because even modest increases in albuminuria predict;progression of renal disease as well as cardiovascular;morbidity (13). Standardization of assays for this;analyte will improve the management of patients with;renal and cardiovascular disease. We previously reported an LC-MS assay for albumin that employed insource fragmentation and had a limit of quantification of;20 mg/L (4, 5). However, because of the prognostic and;diagnostic importance of even modest increases in;urinary albumin excretion within the normal range, we;wanted to improve the limit of quantification (LOQ)6 of;the assay in the normoalbuminuric range (2000 mg/L, n = 38) ranges. All;samples were frozen at -80 C until assayed within 6;months by laborato- rians blinded to the initial result.;The Mayo Clinic In- stitutional Review Board;approved the use of the above specimens.;LIMIT OF QUANTIFICATION AND LIMIT OF DETECTION;The LOQ for urine albumin was calculated as the response for digested HSA spiked into a charcoalstripped urine matrix and resulting in a CV <20%.;The limit of detection was determined using 3 SDs;from the background signal of a zero calibrator digest;without the addition of 15NrHSA.;STATISTICAL ANALYSIS;STUDY POPULATION;Unused human urine samples from those submitted to;the Mayo Clinic Renal Function Laboratory by patients;with clinical indications were acquired and separated;into 3 groups based on the albumin concentration;1102;Clinical Chemistry 55:6 (2009);Regression analysis for method comparisons was performed using linear regression analysis along with;Passing-Bablok regression analysis, which was generated with the statistical program Analyze-it;version;2.12 (Analyze-it Software).;Results;A prerequisite for performing protein cleavage LCMS/MS is selection of peptides that provide;specificity and adequate response to develop a valid;and robust assay. The;tryptic;peptides;LVNEVTEFAK51 and;QTALVELVK534 were chosen on the basis;of exper- imental results from LC-MS/MS;data on tryptic digests from the HSA;calibrators. Several other tryptic peptides of;HSA;were;examined;specifically;13;DLGEENFK20;318;NYAEAK323, 415VPQVSTPTLVEVSR 428;and;546;AVMDDFAAFVEK557.;However, they;lacked ade- quate signal and were not useful;for quantification un- der the conditions;employed.;42;526;Fig. 2. PassingBablok comparison plot of the immunoturbidometric assay vs the LC-MS/MS method using;Representative;the;peptide;chromatograms;LVNEVT transition;for;EFAK y5 (575.5595.6);the;LVNEVTEFAK and;QTALVELVK peptides;along with;their;corresponding;NrHSA peptides;from a 50 mg/L;calibrator;are;provided in Fig. 1.;The retention time;for;the;LVNEVTEFAK;peptide (plots A;and B) was 8.5;15;min, whereas the;QTALVELVK peptide;(plots C and;D) had an elution time;of 9 min.;We determined;the concentration of;albumin in patient;urine samples by;comparing the ratio;of the;urinary albumin to the;peak area to the;NrHSA inter- nal;standard peak area;and referencing to;an;external;calibration curve.;Calibration curves;were determined;using;linear;regression analysis;with;a;1/x;weighting factor on;Analyst software;(Applied;Biosystems).;Representative;calibration curves;are shown for the;LVNEVTEFAK y5ion;and;the;QTALVELVK y5-ion;in;Supplemental;Fig. 1 of the Data;Supplement;that;ac- companies the;15;for all samples ranging;from the;normoalbuminuric;range up to the;macroalbuminuric;range.;Linear regression analysis;(not shown) provided the;follow2;ing relationship: = 0.97.;y = 0.99x +;23.16, R;online version of;this;article;at;http://;www.clinchem.org/;content/vol55/issue;6. Intraassay imprecision;determined;by;using 5 replicates;of low, medium;and high controls;yielded;CVs;<12.4% for both;peptides and their;corresponding ions;(online;Supplemental Table;1).;Interassay;imprecision, determined;by;surveying;calibrators;and;controls for a 10day period, yielded;CVs 30% were observed for certain samples despite the;fact that the LC-MS/MS assay had CVs of approximately 5% in this region. Therefore system error alone;may not be the only factor contributing to the differences observed. For specimens in the macroalbuminuric range (>200 mg/L) the assay displayed a reasonable correlation (Fig. 6A), and percentage differences;were randomly distributed about the mean (Fig. 6B).;However, as with the normoalbuminuric samples, a;slight positive bias was observed for the LVNEVTEFAK;vs the QTALVELVK peptide.;Discussion;Our method for absolute quantification of albumin in;urine by using protein cleavage LC-MS/MS appears to;Clinical Chemistry 55:6 (2009) 1105;Fig. 6. PassingBablok comparison plot for the macroalbuminuric range (>200 mg/L) of the LC-MS/MS;method QTALVELVK;y5;peptide transition;(500.9;587.7) vs the LC-MS/MS method LVNEVTEFAK y peptide transition (575.5595.6).;5;Linear regression analysis (not shown) provided the follow-;= 0.99 (A);and the corresponding BlandAltman plot;over the macroalbumin- uric region (B).;ing relationship, y = 0.97x 36.61, R2;be a viable option for a clinical laboratory setting. This;method provides a lower LOQ compared to an immunoturbidometric system using DiaSorin microalbumin;reagents. This method also provides a high degree of;analytical specificity because the LC-MS/MS methodology incorporates separation and specific mass transitions for the detection of each peptide, and the;LVNEVTEFAK and QTALVELVK peptides are specific;to HSA (accession # P02768, gi:113576). To the best of;our knowledge this is the only report of data obtained;using protein cleavage methodologies and an intact;stable isotope-labeled recombinant internal standard;for quantification of urinary albumin from several;peptides. The previous method reported by our group;using insource fragmentation LC-MS with BSA and;NrHSA internal standards for quantification;af- forded measurement of urinary albumin;without any sample preparation (4, 15).;However, the previous method had a limit;of quantification of 20 mg/L, and thus the;assay performance fell short of analytical requirements needed for a clinical assay (5). To;enhance the quantification of albumin in the;normoalbumin- uric range (<25 mg/L), we;developed a urinary albu- min assay using;protein;cleavage LC-MS/MS. Results;presented here demonstrate that this assay;has an LOQ of 3.13 mg/L, well below the;clinically relevant range, and also has a;large dynamic range (3.13200 mg/L).;15;Because our method incorporates a digestion step;it is theoretically possible that preexisting N-terminal;and/or C-terminal albumin fragments present in urine;could be measured, thereby falsely increasing results;(1719). Truncated versions of albumin have also;been detected in the serum of patients with;pancreatitis (20). To detect such forms in urine, the;ratio of N-terminal and C-terminal albumin peptides;could be compared, with any deviation from unity;suggesting the presence of such preformed N- or Cterminal peptides. How- ever, if the truncations were;minimal it is still possible that these forms could;contain both of the peptides that are monitored in this;assay. In addition, posttransla- tional modifications;within these assayed peptides would change their;masses and therefore cause them to go undetected;thereby falsely lowering the experimen- tal results.;Any modifications to albumin that would inhibit;digestion by trypsin for 2 peptides chosen could also;present falsely low results. Any of the above possibilities could be contributing to the differences observed when results from the 2 peptides were;compared (Figs. 5 and 6). Because the discrepancies;between the albumin peptides we monitored were;relatively minor, our data suggest the majority of;albumin in urine is intact. However, other strategies;and additional studies would have to be developed to;definitively test these possibilities and detect the;presence of these molecules in native urine to;document their potential diagnostic and prognostic;importance. With the above caveats in mind, the;current LC-MS/MS method can serve as a clinical;assay for intact albumin in urine, or more likely as a;reference method.;Urinary albumin quantification is important because increased albumin excretion predicts an increased likelihood of progression to overt renal;disease, and even an increased risk for nonrenal;outcomes (e.g., cardiovascular events). Therefore;efforts to standard- ize this assay are a current priority;following the exam- ple for standardization of serum;1106;Clinical Chemistry 55:6 (2009);creatinine (21). Our results demonstrate that LCMS/MS after trypsin di-;gestion is a viable option for a reference method to;quantify urinary albumin.;Author Contributions: All authors confirmed they have contributed;to the intellectual content of this paper and have met the following 3;re- quirements: (a) significant contributions 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 of Potential Conflicts of Interest: No;authors declared any potential conflicts of interest.;Role of Sponsor: The funding organizations played no role in the;design of study, choice of enrolled patients, review and;interpretation of data, or preparation or approval of manuscript.;Acknowledgments: The authors graciously thank Ward H. Lutz for;NrHSA, Ashia Shaikh for;the help with sample collection, and Sue;Reicks for helpful discussions.;the aid in synthesizing the;15;References;1. Meigs JB, DAgostino RB Sr, Nathan DM, Rifai N;Wilson PW. Longitudinal association of glycemia;and microalbuminuria: the Framingham Offspring;Study. Diabetes Care 2002,25:977 83.;2. Cao JJ, Biggs ML, Barzilay J, Konen J, Psaty BM;Kuller L, et al. Cardiovascular and mortality risk;prediction and stratification using urinary albumin excretion in older adults ages 68 102: the;Cardiovascular Health Study. Atherosclerosis;2008,197:806 13.;3. Mortensen HB, Marinelli K, Norgaard K, Main K;Kastrup KW, Ibsen KK, et al. A nation-wide crosssectional study of urinary albumin excretion rate;arterial blood pressure and blood glucose control;in Danish children with type 1 diabetes mellitus.;Danish Study Group of Diabetes in Childhood.;Diabet Med 1990,7:88797.;4. Babic N, Larson TS, Grebe SK, Turner ST, Kumar R;Singh RJ. 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