Angiotensin I

MALDI-MSI analysis was performed according to our previous study [24 (link)], with minor modifications. The flavan-3-ol standards and strawberry fruit sections were analyzed using a MALDI-TOF/TOF instrument (UltrafleXtreme, Bruker, Billerica, MA, USA) equipped with a 355 nm Nd:YAG laser, using a repetition rate of 1000 Hz. Data were acquired using a step size of 300 μm in negative-ion mode (reflector mode). The m/z values in the range of 240–1200 were measured. The laser diameter was set to the medium size. The instrument was calibrated externally using the exact m/z values of CHCA [M − H]⁻ ions (m/z 188.03532), bradykinin (1–7) [M − H]⁻ ions (m/z 755.38460), angiotensin II [M − H]⁻ ions (m/z 1044.52725), and angiotensin I [M − H]⁻ ions (m/z 1294.67025) as references. The spectra were acquired automatically using FlexImaging 4.1 software (Bruker). Normalization of spectra based on the total ion current was performed using the same software. The FlexImaging 4.1 software was also used to create two-dimensional ion-density maps and for peak analyses.
Matrix screening was performed according to our previous study [25 (link)]. Briefly, 1 µL of each standard (10 μg/mL) was mixed with equal volumes of CHCA (10 mg/mL in 70% aqueous methanol), 9AA (10 mg/mL in 70% aqueous methanol), and DAN (10 mg/mL in 80% aqueous methanol) on an ITO-coated glass slide; then, MALDI-MSI analysis was performed.
To analyze the strawberry fruit sections, frozen sections were took out from a freezer and dried in a vacuum desiccator for 30 min. Six milliliters of a DAN solution (10 mg/mL in 80% aqueous methanol) was sprayed uniformly over the section using a 0.18 mm nozzle caliber airbrush (Mr. Airbrush Custom Double Action; Mr. Hobby, Tokyo, Japan), after which MALDI-MSI analysis was performed. To investigate the spatial distribution of the identified proanthocyanidins, three different strawberry fruits were analyzed. The mass spectra and ion images of the identified flavan-3-ols in the three different strawberry fruits showed similar patterns (Supplementary Figure S3). The mass spectrum and ion images of one of the three different strawberry fruits are presented as representative data in Figure 2C–I.
To compare the intensities of the identified flavan-3-ols between the skin and vascular bundles, three sections of the same strawberry fruit were analyzed and their tissue-specific intensities were obtained using the region-of-interest function of FlexImaging 4.1 software. These data are shown in Figure 5.

Immediately after collection, each sample was centrifuged at 4 °C and placed on ice and stored at −80 °C until the analysis. The urine samples were also immediately stored at −80 °C until the analysis. All the measurements were conducted within 30 days of collection.
After extraction from plasma with alumina, plasma adrenaline was measured by high-performance liquid chromatography (HPLC system JASCO Corporation, Tokyo, Japan) using the procedure described previously by Hunter et al. [9 (link)] with minor modifications. A freezing point depression osmometer (model AUTO&STAT OM-6030, Arkray, Kyoto, Japan) was used to measure the plasma and urine osmolalities. The ion selective electrode method was applied to measure the plasma and urine Na⁺ levels, using an autoanalyzer (BM8060 JEOL Ltd, Tokyo, Japan)Furthermore, the creatininase-creatinase-sarcosine oxidase-POD method was applied for the measurement of plasma and urine creatinine levels, using the above autoanalyzer. The PRA was measured using a method based on the generation of angiotensin I in plasma samples over 60 min at 37 °C followed by the measurement of angiotensin I by a double-antibody ¹²⁵I-radioimmunoassay using a gamma counter instrument (ARC 950,HITACHI Ltd, Tokyo, Japan). A double-antibody ¹²⁵I-radioimmunoassay was applied to measure the plasma ADH using the gamma counter instrument (ARC 950 HITACHI Ltd, Tokyo, Japan), while a competitive solid-phase ¹²⁵I-radioimmunoassay technique was applied to measure Pald, using the kit(SPAC-S Aldosterone kit, TFB inc, Tokyo, Japan).
The following equation was used to calculate the creatinine clearance: (C_Cr mL/min): C_Cr = UCr × V/PCr (UCr: urine creatinine level; PCr: plasma creatinine level; V: urine flow volume). The urinary osmolar excretion was estimated in UosmV mOsm/min, using the formula: UosmV = Uosm × V (Uosm: urine osmolality; V: urine flow volume). The osmolal clearance (in Cosm mL/min) was computed using the equation: Cosm = UosmV / Posm (UosmV: urinary osmolar excretion; Posm: plasma osmolality).
The following formula was used to estimate the free water clearance (in CH2O mL/min): CH2O = V-Cosm (V: urine flow volume; Cosm: osmolal clearance). Finally, we used the following formula to determine the fractional excretion of Na⁺ (FE_Na%): FE_Na = [UNa⁺ × PCr]/[PNa⁺ × UCr] × 100 (PNa⁺: plasma Na⁺; UNa⁺: urinary Na⁺; PCr: plasma creatinine; UCr: urinary creatinine level).

The Ang-I and Ang-II were synthesized, purified, and identified according to the method mentioned in Section 2.5. To a mixture of Ang-I (1 μg) and peptide substrate (1 μg) in 50 µL of borate buffe (200 mM borate buffer, 300 mM NaCl, pH 8.3), 20 µL of ACE (0.05 mU/µL) was added. The mixture was incubated at 37 °C and monitored using LC-MS/MS at 1, 2, 3, 4, 6, and 10 h. The LC separation was performed on Syncronis C18 column (150 mm × 2.1 mm, 5 µm, Thermo Scientific) and eluted with the mobile phase A (5% ACN and 0.1% FA) and B (95% ACN and 0.1% FA) at a flow rate of 350 µL/min. The elution gradient was programmed as (i) 0–5 min, isocratic elution with 5% B; (ii) 5–20 min, linear gradient from 5% to 55% B; (iii) 20–22 min, linear gradient from 55% to 80% solution B; and finally (iv) 22–30 min, isocratic elution maintained at 80% solution B. The degradation of Ang-I and the formation of Ang-II were analyzed using selective ion chromatogram (SIC) on a triple quadrupole mass analyzer (TSQ, Thermo Scientific). The percentage of the remaining angiotensin I (RA%) at each time point was calculated as A_I/(A_I + A_II) × 100%, where A_I is the SIC peak area of remaining Ang-I and A_II is the peak area of the resulting Ang-II.