Flavonoids Interference in Common Protein Assays: Effect of Position and Degree of Hydroxyl Substitution

Abstract

Flavonoids interferes with colorimetric protein assays in a concentration- and structure-dependent manner. Degree (≥3) and position (C₃) of -OH substitution was associated with intensified interference (p<0.05). Significant overestimation of protein (~3–5 folds) could occur at higher flavonoid concentrations (>5μM) and is particularly evident at lower protein concentrations (25–250 μg/ml). Since, healthy human urinary protein (< 200 μg/ml) and flavonoids urinary excretion (0.5–2 μg/ml) levels fall in this range, overestimation of protein concentration with flavonoids consumption in diet, including natural supplements, remains relevant issue for diagnostic and research labs. Protein precipitation by acetone to remove interfering flavonoid successfully resolve the problem.

1. Introduction

Protein estimation is a regular experimental protocol for many in vitro cellular and in vivo tissues studies. Bicinchoninic acid (BCA) protein assay is a simple and accurate method to measure the concentration of solubilized proteins in biomedical research^1,2. Pierce™ BCA assay uses the principle of reduction of Cu⁺² to Cu⁺¹ in an alkaline solution (the biuret reaction), which then interacts with BCA to give purple color^3,4 that absorbs at 562 nm and can be quantified using spectrophotometer. Another copper reduction-dependent assay is Lowry assay (Bio-rad DC), where the color formation is a two-step process, reaction of protein with an alkaline copper tartrate solution, and subsequent reduction of Folin Phenol reagent by copper-treated protein^5,6. Both assays are frequently used in research labs for cellular, tissue and urine protein concentrations and for clinical diagnostic purposes including proteinuria tests^7–10.

In drug development ADME studies, protein analysis commonly used in in vitro drug absorption, metabolism and transport cell-based studies as well as tissue protein concentrations in pharmacokinetic studies. In cellular fraction metabolism studies, protein analysis is usually done prior to incubation with drugs, however in most other studies, protein concentration of cells or tissues are determined after the termination of experiment. It is very common for drugs and/or their metabolites to accumulate inside cells¹¹, which can make protein concentration determination challenging, if the drug and/or metabolite act as interfering agent in the protein assay.

In literature, reducing agents (such as DDT and EDTA) has been mentioned as a source of interference in BCA and Lowry protein analysis^2,3. Therefore, it is not unlikely that xenobiotics with antioxidant properties, such as flavonoids, could also interfere with these assays. Flavonoids are the polyphenolic compounds occurring naturally in variety of fruits and vegetables, and beverages such as tea and wine. Flavonoids have shown to have various health benefits such as antioxidant, cancer chemoprevention, relief from post-menopausal systems, prevention against aging disorders, neurodegenerative and cardiovascular diseases¹². According to Nurse’s Health Study and National Health and Nutrition Examination Survey, estimated daily flavonoids intake in United states has been reported as ~200–400 mg^13,14, whereas daily urinary excretion of flavonoids (including phase-II metabolites) with that kind of intake can range between 0.5–2 μg/ml¹⁵. The antioxidant potential of flavonoids is due to the presence of multiple hydroxy in their structure, which however also makes flavonoids susceptible to rapid chemical degradation due to oxidation in aqueous medium.

Though Bradford protein assay lists flavonoids as one of the interfering agents^16–18, so far no systemic study have been published to ascertain the extent of interference of flavonoids in commonly used biochemical protein assays, such as BCA and Lowry, at their physiologically relevant cellular or urinary concentrations. We believe that with increasing interest in studying the various beneficial effects of flavonoids and other polyphenolic compounds in research labs and significant dietary intake of flavonoids worldwide, it is important to understand their inference in the protein determination assay. In this study, we investigated various flavonoids and their metabolites as a source of interference in two commonly used protein assays.

2. Materials and Methods

2.1. Materials

A total of 54 flavonoids and related compounds were used for the study (Table S1). Apigenin-7-O-glucuronide was purchased from HWI Analytik GmbH (Rheinzaberner, Rülzheim, German). Quercetin-3-O-glucuronide, luteolin-3’-O-glucuronide, luteolin-7-O-glucuronide, wogonin, luteolin were purchased from Meilunebio (Dalian, China). Raloxifene, raloxifene-4’-Oglucuronide, raloxifene-6-O-glucuronide were from Toronto Research Chemical. All other Most flavonoids used in the study were purchased from Indofine Chemicals (Somerville, NJ). Pierce™ BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA). Bio-rad DC assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). Precision Labs Protein Test Strips was purchased from Precision Laboratories, Inc (Cottonwood, AZ).

2.2. Protein Standard and Flavonoid Working Solutions

Standard solutions of BSA with concentrations 25, 125, 250, 500, 750, 1000, and 1500 μl were prepared with appropriate dilution of standard solution (2 mg/ml) with purified water. 500, 250, 50, 25, 5, and 2.5μM working solutions of the tested flavonoids were prepared in 50% MeOH solution by diluting 5, 10 or 50 mM stock solutions in DMSO:Ethanol (1:9).

2.3. Protein Assay

BCA or Bio-rad protein assay standard curves was prepared as per respective protocols^3–5 with the following modification. 4.7 μl of compound solution (at 500, 250, 50, 25, 5 or 2.5 μM concentration) was added in each well after the protein standard before adding colorimetric reagents to provide approximate final compound concentrations of 10, 5, 1, 0.5, 0.1 or 0.05 μM, respectively. 50% MeOH was added for the preparation of blank standard curves. Purified water was used in place of protein standards while determining the baseline absorbance of flavonoids.

2.4. Protein Precipitation

Protein precipitation to remove interfering substances in BCA assay was done as described in literature using Trichloro acetic acid (TCA) or acetone¹⁹ in presence of 10μM of either very interfering flavonoids (3,3’,4’-trihydroxyflavone or quercetin) or non-interfering flavonoids (5,4’-dihydroxyflavone or wogonoside). Procedure flowchart has been showed in supplementary material, Figure S13. Additionally, acetonitrile was tried instead of acetone using the same procedure. In one case, cold diethyl ether was added to remove extra TCA before reconstitution of protein. Protein standard curve were prepared after protein reconstitution in each method and loss of protein was calculated for each method.

2.5. Protein Strip Test

Protein strip dip test was conducted using 4 standard protein concentrations and 50, 250 and 500 μM of known non-interfering (7-hydroxyflavone) and interfering flavonoids and glucuronides (quercetin, quercetin-3-O-glucuronides, epicatechin and 3,7, 3’-trihydroxyflavone) as per manufacturer’s instruction. Protein strips were used to perform dip test using 0.25 mg/ml and 0.5 mg/ml BSA solution in water and 0.1% w/v Vitamin C (ascorbic acid) solution (pH adjusted to 7) with and without quercetin (at 0.1, 1, 5, and 10μM) and quercetin-3-O-glucuronide (5 and 10μM).

2.6. Statistical Analysis

The data in this report was analyzed either using non-parametric Wilcoxon matched-pair test, or unpaired non-parametric Mann-Whitney Test and one-way ANOVA, no pairing, non-parametric Kruskal-Wallis test using GraphPad Prism version 8.

3. Results and Discussion

Most flavonoids and their glucuronides can significantly interfere with BCA and Lowry protein assays in a concentration- and structure- dependent manner (Figures 1 and 2), the effect significantly more relevant at lower protein concentration range. The proposed mechanism of interference is due to the reduction of Cu⁺² to Cu⁺¹ by flavonoids, which then interacts with BCA (or Folin Phenol reagent) to give different intensity of purple (or green color), based on flavonoid structure and concentration present, even in the absence of protein (Figure 1).

Figure 1.

Heat-map of baseline absorbance of different flavonoids at 1, 5 and 10 μM using Pierce™ BCA assay @ 562 nm (left panel) and Bio-rad Lowry assay at 750 nm (right panel) in absence of protein.

Figure 2.

Effect of flavonoid concentrations and number of hydroxyl groups in flavonoid structure on the inference in Pierce™ BCA (A, B) and Bio-rad Lowry (B, D) methods. Baseline absorbance of 54 flavonoids (7 with one hydroxyl, 15 with two hydroxyl, 13 with three hydroxyl and 7 with more than 3 hydroxyl substitution) at 1, 5 and 10 μM were compared at different concentrations. Wilcoxon matched-pair test was used for statistical analysis for A and B, and unpaired non-parametric Mann-Whitney test (black and red color) and one-way ANOVA no pairing, non-parametric Kruskal-Wallis tests (green color) were used for C and D ( **** p< 0.0001; *** p<0.001; ** p<0.01; ns not significant).

In BCA protein assay, 1 μM compound concentration overestimated protein concentration in range of 25–250 μM, whereas 5 or 10 μM compound concentration overestimated protein concentration over the whole range of standard curve (25–1500 μM) for certain compounds such as quercetin (Supplementary Materials, Figure S1-S2). For example, the protein values in the presence of 10μM quercetin at 125 μg/ml, 500 μg/ml and 1000 μg/ml were overestimated approximately by 390%, 96% and 60%, respectively. Whereas, in presence of 1 μM quercetin, overestimation of 125 μg/ml, 500 μg/ml and 1000 μg/ml protein was about 150%, 56% and 20%, respectively. The interference was found to be significant for both protein assays, but the extent of interference was dependent on flavonoid-protein assay combination used for many compounds.

The overestimation of BSA protein at lower concentration (250 μg/ml) in the presence of 11 tested flavonoids (at 10μM) was in the similar range for both BCA (~ 50–170%) and Bio-rad protein assays (~ 25–300%) (Supplementary Materials, Figure S3), however, the rank order of interference in the two protein assays differed, such that certain flavonoids with lower inference in BCA showed significant inference in Bio-rad and vice a versa, for e.g. quercetin and naringenin (Figure 1 and Supplementary Materials, Figure S3, Table S1). The backbone structure as well as degree and position(s) of hydroxyl group substitution in the structure of flavonoids were the determining factor for the extent of interference in the protein assay (Supplementary Materials, Figures S4 and S5). The increase in degree of hydroxyl groups substitution in general increased the level of interference up to 3 hydroxyl groups (Figure 2), the effect more visible at higher flavonoid concentrations (Supplementary Materials, Figure S6). The interference was especially prominent with flavonol subclass of flavonoids, where a hydroxyl group must be present at C₃ position on the flavonoid scaffold for BCA assay (Supplementary Materials, Figure S7).

Flavonol subclass constitutes the major percentage of the estimated dietary intake of flavonoids in United States population, and were found to be most prone to oxidative degradation (based on in-house determination of stability), however the relative position of multiple hydroxyl group(s) in the structure did affect the rate of degradation. Among monohydroxyflavone and dihydroxyflavones, flavanols showed significantly higher interference in the protein analysis. Among all tested trihydroxyflavones, 3,3’,4’-trihydroxyflavone and 3,6,4’-trihydroxyflavone showed significantly higher (2-folds higher than blank) absorbance in BCA analysis (Figure 1, and Supplementary Materials, Table S1).

In a previously published report of structure-antioxidant property of flavonoids, Silva et al. (2002) showed that hydroxyl substitution (-OH) at C₃, 2,3-double bond and presence of ortho-catechol group (-OH group at 3’ and 4’) in the B-ring in flavonoid structure contributes to higher antioxidant activity²⁰. Results in the present study aligned somewhat with these observations. There was significant difference (p<0.0001) between interference due flavonoids with and without C₃ hydroxyl substitution in BCA assay but no difference in Bio-rad assay (Supplementary Materials, Figure S7). Also, flavonoids with ortho-catechol group in A or Bring, such as, 3,3’,4’-trihydroxyflavone, quercetin, luteolin, and epicatechin, showed the higher interference in BCA protein analysis (Supplementary Materials, Table S1). However, the effect was weak or not significant when all compounds with catechol group were compared with all compounds with no catechol group in BCA and Bio-rad assay, respectively (Supplementary Materials, Figure S8).

Since, glucuronides are one of the major forms of dietary flavonoids excreted in urine and accumulated in liver and intestinal cells assays, we also studied the impact of glucuronidation on the interfering potential of certain flavonoids. Though there was no specific pattern, as interference either remained same, increased or decreased based on aglycone structure and position of O-glucuronidation, data showed that glucuronidation in most cases did not eliminate the inferring potential of flavonoids (Supplementary Materials, Figures S9 and S10).

We investigated the commonly used method of protein precipitation by different reagents, such as trichloroacetic acid (TCA), trichloroacetic acid + diethyl ether (TCA + DiEE), acetone or acetonitrile (ACN) using two most interfering flavonoids (quercetin and 3,3’,4’trihydroxyflavone) and two least interfering flavonoids (5,4’-dihydroxyflavone and wogonoside or wogonin-7-glucuronide).

Results showed that precipitation with acetone was the best method to remove flavonoids interference without losing too much protein, though protein loss was higher at lower concentrations (Figure 3 and Supplementary Materials, Figures S11 and S12, Table S2). TCA was found to be good at protein recovery at lower concentrations but performed very poorly at higher protein concentration. Addition of diethyl ether to remove extra TCA resulted in further loss of protein (Figure 3A, and Supplementary Materials, Table S2). Precipitation with ACN was also found to be a poor method (Figure 3B). It is to be noted that acetone though shown to be a better precipitating agent than TCA in this study, may still cause some loss of protein, depending upon the protein concentration, type of proteins precipitated, pellet solubilization buffer and sonication²¹.

Figure 3.

Comparison of various protein precipitation reagents using BCA method in presence of 10μM one of the most interfering flavonoids, quercetin; Quer (A) and one of the least interfering flavonoids, 5,4’-dihydroxyflavone; 5,4’-DHF (B). Standard curves of BSA protein were prepared in absence of flavonoid (blank, n=2), presence of 10 μM flavonoid (Quer or 5,4’-DHF, n=1), and protein precipitation with tricholoracetic acid (+ TCA ppt, n=3), tricholoracetic acid followed by diethyl ether (+ TCA + DiEE ppt, n=3), acetonitrile (+ CAN ppt, n=3) and acetone ( + acetone ppt, n=3). Linear regression was plotted for each data set and equation and regression coefficient r² values are shown below the graph.

The semi-quantitative protein strip dip test showed immediate color development with flavonoids and glucuronides solutions at higher concentration (50, 250, 500μM) when reading after 60 minutes time as indicated in the manufacturer’s instruction. The color development corresponded to 0.25–0.5 mg/ml BSA protein. However, the color slowly faded away as the time lapsed (Supplementary Materials, Figures S14 A-D). We believed the loss of color with time was probably related to the loss of the oxidative ability of flavonoids over time, therefore 0.1% vitamin C was added to the solution to protect the oxidative ability of quercetin and quercetin-3-O-glucuronide at the lower concentrations. No visibly measurable interference with either quercetin and quercetin-3-O-glucuronide at any of the tested concentrations (0.1, 1, 5 and 10 μM) in water or 0.1% vitamin C solution was observed at the tested BSA protein concentrations (0.25 mg/ml and 0.5 mg/ml) from observation made up till 8 minutes post-dipping (Supplementary Materials, Figures S15 A-B). These results indicated that the protein overestimation in semi-quantitative protein strip dip test may not be visible if the flavonoids (and/or their glucuronides) are not present at high concentrations in urine samples, and a wait time of at least >5 min is needed before the reading is taken. We suggest that the interference potential of flavonoids in urine should be tested and wait time optimized for any protein strip product in the market prior to clinical use.

However, it is to be noted that more sensitive quantitative analysis of protein in case of chronic kidney disease will lead to significant overprediction of disease severity in the presence of dietary flavonoids excreted in the urine, mostly as glucuronides. The normal protein concentration range found in healthy human urine generally is less than 75–200μg/ml (~ <150 mg/day), whereas in the cases of chronic kidney disease, urinary protein excretion can be as high as 1000–2500 μg/ml (~ 2 g/day), based on the daily urinary output^22,23. Our present study strongly indicates that presence of certain flavonoid at a concentration as low as 10μM in urine can significantly overestimate the normal protein levels (~200 μg/ml) to those found in disease state (~1000 μg /ml), therefore giving false-positive results for chronic kidney diseases. We suggest that in case of high protein levels found in urine, flavonoids interference needs to be ruled out before other clinical tests for the presence of kidney disease are considered.

4. Conclusion

Most commonly used flavonoids and their glucuronides/glucoside in diet including dietary supplements and preclinical research can significantly interfere with commonly used protein assays, especially in low protein concentration range that is usually found in healthy human urine. Since effective interfering concentrations of flavonoids and glucuronides can be easily achieved intracellularly (> 1 μM) in in vitro and animal studies, protein analysis in the presence of flavonoids can easily lead to overestimation (by ~3–5 folds) of protein. Such overestimation in urine can lead to misdiagnosis of proteinuria, wrongly indicating presence of kidney disease⁷.Also, protein overestimation could cause underestimation of enzymatic activity in in vitro assays. In event of moderate to high dietary or supplemental intake of flavonoids in an individual’s diet, the urine protein analysis in diagnostic lab/clinics should be performed using either appropriate precipitation method to remove interfering substances or semi-quantitative strip dip test should be used after appropriate testing for interference and optimization of wait time before reading.

Highlights.

• Flavonoid Interference in BCA and Lowry Protein Assay has been systematically demonstrated using structurally different flavonoids.

• Report discusses the implications of dietary flavonoids and their glucuronides excreted in Urine on overestimation of proteinuria test results.

• Several protein precipitation solvents are evaluated for dealing with flavonoid interference in research and diagnostic labs in different protein concentration range.