Application of Vitamin C Measurement Procedure using 4-Hydroxy-2, 2, 6, 6-Tetramethylpiperidine-1-Oxyl and O-Phenylenediamine Dihydrochloride for Various Fruits

Abstract

Application of a measurement procedure for total Ascorbic Acid (AsA) content (i.e., the sum of AsA and Dehydro Ascorbic Acid (DAsA)) in various fruits using 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-1-oxyl and o-phenylenediamine dihydrochloride was introduced. Total AsA and DAsA contents in 13 fruits (i.e., kiwifruit, pineapple, mango, tomato, grapefruit, blueberry, banana, watermelon, plum, peach, melon, apple and pear) were measured, and the observed total AsA content in those fruits was significantly correlated with that reported in the “Standard Tables of Food Composition in Japan”.

Keywords: Nutrition; Ascorbic acid; Vitamin C; Fruits; 4-Hydroxy-2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO)

Abbreviation

AA: Ascorbic Acid; DHAA: Dehydroascorbic Acid; SVCT1: Sodium-dependent Vitamin C Transporter Type 1; GLUT1: Glucose Transporter 1; TEMPO: 4-Hydroxy-2, 2, 6, 6-tetramethylpiperidine- 1-oxyl; OPDA: o-Phenylenediamine Dihydrochloride; MPA: MetaphosphoricAcid; HPLC: High-performance Liquid Chromatography; ECD: Electrochemical Detection; DNPH: 2, 4-Dinitrophenylhydrazine; GLO: L-GulonolactoneOxidase; RDA: Recommended Dietary Allowance

Introduction

We previously developed an automated method for measuring total ascorbic acid concentration (i.e., the sum of ascorbic acid: AsA and dehydro ascorbic acid: DAsA) in serum using 4-hydroxy-2, 2, 6, 6-Tetramethylpiperidine-1-Oxyl (TEMPO) and O-Phenylenediamine Dihydrochloride (OPDA) [1]. OPDA reacts exclusively with DAsA, and the AsA in specimens was first oxidized to DAsA by the addition of TEMPO. DAsA originally present in the specimens and that derived from As A were both condensed with OPDA to form a fluorescent product measurable by spectrophotometry at 340 nm. Good agreement had been shown in total AsA concentrations in serum between measurement by the TEMPO method and that by High-Performance Liquid Chromatography (HPLC) with Electro Chemical Detection (ECD) [TEMPO = 0.99 (HPLC) + 0.1, r = 0.981]. Laboratories without access to HPLC could measure serum total AsA concentration using the TEMPO method.

Previously, we applied the automated TEMPO method for total AsA and DAsA concentrations in urine specimens [2]. Furthermore, we reported that urinary excretion of DAsA in healthy adults varied from 0.9 to 13.8 mg/L. Here, we introduce the possibility of application of the TEMPO method [3] for measurement of total AsA and DAsA contents in various fruits. The present method is a manual procedure, and is thus suitable for laboratories without access to an automated analyzer.

Sample treatment: Ten volumes of 4.0% Meta Phosphoric Acid (MPA) were added to one part flesh of the fruits (some contained the peel) or squeezed juice of the fruits (w/v). After homogenization, samples were centrifuged at 6,200 rpm (2,000 × g) for 5 min to spin down the pellet and obtain a clear supernatant. The supernatant was used for the following analysis.

Materials and Methods

Reagent

TEMPO solution was prepared by dissolving 20 mg of TEMPO (Sigma-Aldrich Co. LCC., St. Louis, MO, USA) in 100mL of 0.067 mol/L phosphate buffer, pH 6.4. OPDA solution was prepared by dissolving 5 mg of OPDA (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in 10mL of the same buffer, followed by storage in a refrigerator (4°C) in the dark. MPA solution (4%) was prepared by dissolving 4 g of potassium metaphosphate (Wako Pure Chemical Industries) in 100 Ml of distilled water. A standard solution was prepared by dissolving 10 mg of DAsA (Wako Pure Chemical Industries) in 100mL of distilled water.

Assay procedure

An aliquot (0.125mL) of the above supernatant (“measurement”) or standard solution (“calibration”) was mixed with 1.0 mL of TEMPO solution and incubated at 37°C for 5 min. Next, 0.425 mL of OPDA solution was added to the mixture, and the absorbance (A340) of the reaction mixture was measured using a Hitachi 7012 spectrophotometer (Hitachi High-Technologies Corporation, Tokyo, Japan) for 15 min. AsA content (mg/L) in the supernatant was obtained from the A 340 of “measurement” as compared with that of “calibration”, and AsA content in fruits was expressed in terms of mg AsA per 100 g of fruit. For the measurement of DAsA, TEMPO solution was replaced by 0.067 mol/L phosphate buffer, pH 6.4, followed by incubation and addition of OPDA solution in the same manner as with the AsA measurement in the absence of TEMPO. The assay range was linear from 0.0 mg/L up to 100 mg/L for DAsA content, but the Limit of Quantitation (LOQ) was 1.0 mg/L. Despite being a manual procedure, within-day variation for AsA content (10.0±0.2mg/L) was found to have a Coefficient of Variation (CV) of 2.0%. Between-day variation for the same samples over ten successive days exhibited a CV of 4.7%. Meanwhile, pressed apple juice spiked with 10.0 mg/L AsA showed a recovery of 13.1 mg/L.

Total AsA and DAsA contents in the various fruits were assayed (Table 1). Although not the same sample was assayed, total AsA content in these fruits (except for melon) was significantly correlated with those reported in the “Standard Tables of Food Composition in Japan: STFCJ [4]” [TEMPO = 1.08 (STFCJ) + 0.04, r = 0.970]. However, conflicting results were observed with regard to DAsA content between our study and previous reports. In our study, DAsA and AsA contents [(DAsA, mg/100g)/ (total AsA, mg/100 g)] were 7.8/8.4 (93%) in banana and 4.0/4.3 (92%) in apple. In contrast, Mazurek et al. [5] reported these respective contents as 2.24/15.42 (14.5%) in banana, while Roe et al. [6] reported 0.0/4.5 (0.0%) in apple; all of the samples were purchased from a city market. However, Wills et al. reported that DAsA content was not present in fresh banana fruits assayed following purchase from local retail markets in Australia [7].

  

    

    
Our analysis
    
STFCJ
  
  

    
Total AA (mg/100 g)
    
DHAA (mg/100 g)
    
DHAA/Total AA ratio (%)
    
Total AA (mg/100 g)
  
  

    
Apple
    
18.1
    
6.3
    
34.8
    
6
  
  

    
Banana
    
10.0
    
9.4
    
94.0
    
16
  
  

    
Dragon fruit
    
3.4
    
2.9
    
85.3
    
7
  
  

    
Grapefruit
    
40.6
    
9.6
    
23.6
    
36
  
  

    
Japanese lemon
    
53.4
    
8.3
    
15.5
    
40
  
  

    
Kiwifruit
    
101.6
    
13.0
    
12.8
    
69
  
  

    
Lemon
    
52.7
    
9.4
    
17.8
    
50
  
  

    
Mango
    
18.6
    
3.2
    
17.2
    
20
  
  

    
Oleaster
    
8.1
    
7.2
    
88.9
    
5
  
  

    
Pineapple
    
59.6
    
10.8
    
18.1
    
27
  
  

    
Satsuma mandarin
    
46.9
    
7.2
    
15.4
    
32
  
  

    
Strawberry
    
78.3
    
11.3
    
14.4
    
62
  
  

    
AsA:    Ascorbic Acid; DAsA: Dehydro Ascorbic Acid; STFCJ: Standard Tables of Food    Composition in Japan (2015).  
  

Table 1: AsA and DAsA contents in various fruits.