Methods and Systems for Measuring Plasma Renin Activity

Abstract

Disclosed are methods and systems for the quantification of AngI and/or determination of plasma renin activity in a sample. The methods and systems disclosed herein can be useful for diagnosis of hypertension, aldosteronism and other abnormalities of the renin angiotensin aldosterone system (RAAS).

We claim: 
     
         1 . A method for determining an amount of AngI in a sample, the method comprising:
 (a) incubating the sample under conditions suitable to allow plasma renin to generate angiotensin I (AngI) of a sequence NH2-DRVYIHPFHL-COOH (SEQ ID NO:1) from angiotensinogen present in the sample;   (b) adding to the sample a protease or a chemical hydrolyzing agent capable of hydrolyzing AngI to generate a defined AngI cleavage product;   (c) incubating the sample under conditions to allow the protease or the chemical hydrolyzing agent to hydrolyze the AngI to generate the defined AngI cleavage product;   (d) ionizing the defined AngI cleavage product to produce one or more AngI cleavage product ions detectable by mass spectrometry; and,   (e) detecting the one or more AngI cleavage product ions by mass spectrometry to determine the amount of AngI in the sample.   
     
     
         2 . The method of  claim 1 , further comprising adding a stable isotope-labeled angiotensin I peptide (SIL-AngI) to the sample as an internal standard. 
     
     
         3 . The method of  claim 2 , wherein the SIL-AngI is a peptide of a sequence NH2-DRV{circumflex over ( )}YIHP{circumflex over ( )}F{circumflex over ( )}HL-COOH (SEQ ID NO: 3), wherein {circumflex over ( )} indicates an amino acid labeled with a heavy isotope. 
     
     
         4 . The method of  claim 3 , wherein V{circumflex over ( )} is (13C)5H11(15N)O2, having a mass shift of +6; P{circumflex over ( )} is (13C)5H9(15N)O2, having a mass shift of +6; and F{circumflex over ( )} is (13C)9H11(15N)O2, having a mass shift of +6. 
     
     
         5 . The method of  claim 2 , wherein the SIL-AngI peptide comprises amino acids labeled with a heavy isotope. 
     
     
         6 . The method of  claim 2 , wherein the SIL-AngI peptide is added before or after step (a), but prior to step (b), and the method further comprises generating an SIL-AngI cleavage product by hydrolysis by the protease or the chemical hydrolyzing agent, and ionizing the SIL-AngI cleavage product to generate SIL-AngI cleavage product ions. 
     
     
         7 . The method of  claim 6 , wherein an amount of AngI cleavage product is directly proportional to an amount of the SIL-AngI cleavage product. 
     
     
         8 . The method of  claim 1 , further comprising generating a plurality of calibration standards comprising known amounts of AngI in a suitable matrix, wherein each of the plurality of the calibration standards comprises AngI of SEQ ID NO: 1 that is subjected to the same steps as the sample AngI. 
     
     
         9 . The method of  claim 8 , wherein the amount of AngI in the sample is quantified by comparing an amount of the one or more AngI cleavage product ions detected in the sample to an amount of one or more AngI cleavage product ions detected in at least one of the plurality of calibration standards. 
     
     
         10 . The method of  claim 9 , wherein the calibration standards comprise AngI concentrations ranging from 0.25 to 100 ng/mL. 
     
     
         11 . The method of  claim 1 , wherein the protease is a serine protease. 
     
     
         12 . The method of  claim 1 , wherein the protease is trypsin. 
     
     
         13 . The method of  claim 1 , wherein the chemical hydrolysis agent is formic acid, acetic acid, or hydrochloric acid. 
     
     
         14 . The method of  claim 1 , wherein the hydrolysis by the protease or the chemical hydrolyzing agent generates an AngI cleavage product of a sequence NH2-VYIHPFHL-COOH (SEQ ID NO:2). 
     
     
         15 . The method of  claim 3 , wherein the SIL-AngI peptide is added before or after step (a), but prior to step (b), and wherein hydrolysis by the protease or the chemical hydrolyzing agent generates a SIL-AngI cleavage product of a sequence NH2-V{circumflex over ( )}YIHP{circumflex over ( )}F{circumflex over ( )}HL-COOH (SEQ ID NO:4). 
     
     
         16 . The method of  claim 1 , wherein the one or more AngI cleavage product ions are selected from the group consisting of ions having a mass/charge ratio of 513.3±2, 269.2±2, 392.7±2, and 257.1 ±2. 
     
     
         17 . The method of  claim 7 , wherein the SIL-AngI cleavage product ions are selected from the group consisting of ions having a mass/charge ratio of 524.3±2, 779.5±2, and 269.2±2. 
     
     
         18 . The method of  claim 1 , further comprising terminating plasma renin activity prior to protease digestion. 
     
     
         19 . The method of  claim 18 , wherein methanol is added to the sample to terminate plasma renin activity. 
     
     
         20 . The method of  claim 19 , further comprising evaporating the methanol and then reconstituting the sample in a buffer prior to the protease digestion. 
     
     
         21 . The method of  claim 1 , wherein the defined AngI cleavage product is subjected to liquid chromatography prior to mass spectrometry. 
     
     
         22 . The method of  claim 1 , wherein the ionizing is performed by positive electrospray ionization with selected reaction monitoring (SRM). 
     
     
         23 . The method of  claim 1 , wherein the sample is a biological fluid obtained from a patient. 
     
     
         24 . The method of  claim 23 , wherein the biological fluid is plasma. 
     
     
         25 . The method of  claim 24 , wherein the biological fluid comprises ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. 
     
     
         26 . The method of  claim 1 , further comprising, prior to step (a), removing an aliquot from the sample and testing the aliquot for presence of EDTA. 
     
     
         27 . The method of  claim 26 , wherein the testing for the presence of EDTA comprises addition of a colorimetric agent, o-cresolphthalein complexone, to react with calcium ions in the sample, such that samples comprising EDTA remain substantially unchanged in color, whereas samples that do not have EDTA turn color due to reaction of calcium with the colorimetric agent. 
     
     
         28 . The method of  claim 1 , wherein level of plasma renin activity in the sample is calculated based on the amount of AngI in the sample. 
     
     
         29 . The method of  claim 1 , wherein the plasma renin activity is defined by the amount of AngI generated per unit time. 
     
     
         30 . The method of  claim 29 , wherein the plasma renin activity is expressed as ng/mL/hr. 
     
     
         31 . The method of  claim 30 , wherein the plasma renin activity has an analytically measurable range (AMR) ranging from about 0.167-66.667 ng/mL/hr. 
     
     
         32 . The method of  claim 30 , wherein a lower limit of quantification (LLOQ) of the plasma renin activity is 0.167 ng/mL/hr and an upper limit of quantification (ULOQ) of the plasma renin activity is 66.667 ng/mL/hr. 
     
     
         33 . A method for determining plasma renin activity (PRA) in a sample, the method comprising:
 (a) incubating the sample under conditions suitable to allow plasma renin to generate angiotensin I (AngI) of a sequence NH2-DRVYIHPFHL-COOH (SEQ ID NO:1) from angiotensinogen present in the sample;   (b) adding to the sample a protease or a chemical hydrolyzing agent capable of hydrolyzing AngI to generate a defined AngI cleavage product;   (c) incubating the sample under conditions to allow the protease or the chemical hydrolyzing agent to hydrolyze the AngI to generate the defined AngI cleavage product;   (d) ionizing the defined AngI cleavage product to produce one or more AngI cleavage product ions detectable by mass spectrometry; and,   (e) detecting the one or more AngI cleavage product ions by mass spectrometry, wherein detected amount of the one or more AngI cleavage product ions is indicative of level of the PRA in the sample.   
     
     
         34 . The method of  claim 33 , wherein the protease is a serine protease. 
     
     
         35 . The method of  claim 33 , wherein the protease is trypsin. 
     
     
         36 . The method of  claim 33 , wherein protease cleavage generates an AngI cleavage product of a sequence NH2-VYIHPFHL-COOH (SEQ ID NO:2). 
     
     
         37 . The method of  claim 33 , further comprising:
 adding a stable isotope-labeled angiotensin I peptide (SIL-AngI) to the sample as an internal standard before or after step (a), but prior to the step (b);   in step (c), incubating the sample to allow the protease or the chemical hydrolyzing agent to hydrolyze an internal standard to generate an internal standard cleavage product;   in step (d), ionizing the internal standard cleavage product to generate internal standard cleavage product ions; and,   in step (e), detecting the AngI cleavage product ions and the internal standard cleavage product ions to determine an amount of the AngI cleavage product and an amount of the internal standard cleavage product in the sample.   
     
     
         38 . The method of  claim 37 , wherein the SIL-AngI is a peptide of a sequence NH2-DRV{circumflex over ( )}YIHP{circumflex over ( )}F{circumflex over ( )}HL-COOH (SEQ ID NO:3), wherein {circumflex over ( )} indicates an amino acid labeled with a heavy isotope. 
     
     
         39 . A method for determining plasma renin activity (PRA) in a sample, the method comprising:
 (a) incubating the sample under conditions suitable to allow plasma renin to generate angiotensin I (AngI) of a sequence NH2-DRVYIHPFHL-COOH (SEQ ID NO:1) from angiotensinogen present in the sample;   (b) before or after step (a), but prior to step (c), adding a stable isotope-labeled angiotensin I peptide (SIL-AngI) to the sample as an internal standard;   (c) adding to the sample a protease or a chemical hydrolyzing agent capable of hydrolyzing AngI to generate a defined AngI cleavage product;   (d) incubating the sample under conditions to allow the protease or the chemical hydrolyzing agent to hydrolyze the AngI to generate the defined AngI cleavage product, and to allow the protease to hydrolyze the SIL-AngI peptide to generate an SIL-AngI cleavage product;   (e) ionizing the defined AngI cleavage product to produce one or more AngI cleavage product ions detectable by mass spectrometry, and ionizing the internal standard cleavage product to generate one or more SIL-AngI cleavage product ions detectable by mass spectrometry; and,   (f) detecting the one or more AngI cleavage product ions and the one or more SIL-AngI cleavage product ions to determine an amount of the AngI cleavage product and an amount of the SIL-AngI cleavage product in the sample,   wherein a ratio of the determined amounts of the AngI cleavage product and the internal standard cleavage product is indicative of an amount of PRA in the sample.