Guidelines for purine extraction and determination in foods

Abstract

With the improvement of living standards over the years, the demand for “delicious food” has been increasing rapidly. Accordingly, the hidden risks associated with these kinds of food have gradually been attracting attention and purines have especially become of great interest. Food‐derived purines are one of the important sources of purine in the body. Eventually, it is metabolized by the body to produce uric acid, which may lead to hyperuricemia and gout. Thus, detecting the purine content in foods could be a significant guidance for dietary intake. Here, this article reviews purine pretreatment and detection methods as well as the purine content of common foods. Perchloric acid hydrolysis and high‐performance liquid chromatography were found to be the most commonly used pretreatment and quantification methods in purine detection. In addition, total purine content was in the order of plant foods < fungus and algae foods < animal foods < aquatic products. This reenforces the suggestion that, for individuals with hyperuricemia and gout, fewer or none intake of aquatic products and animal foods and increase intake of plant and fungal and algae foods may be a healthier option.

INTRODUCTION

Purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Two of the bases in nucleic acids, adenine, and guanine, are purines. Purines in foods mainly exist in the combined form of nucleotides, nucleosides, and bases. Upon digestion, they are hydrolyzed into adenine and guanine. A portion is used by the body to synthesize DNA and RNA while the remaining portion is further degraded to produce xanthine and hypoxanthine (the structures of adenine, guanine, xanthine, and hypoxanthine are shown in Figure 1). As can be seen from Figure 2, one-third of uric acid is produced exogenously and two-third is produced endogenously. Uric acid is excreted mainly through renal filtration (around 90% is reabsorbed through renal tubules and 10% is excreted in the form of urine). Therefore, the human body normally maintains the balance of uric acid absorption and excretion (Benn et al., 2018). Subsequent catalysis by xanthine oxidase eventually produces uric acid (Figure 3) (Kaneko et al., 2014). Besides, the produced uric acid is released into the bloodstream and is eventually filtered by the kidneys and excreted in the urine. In nonhuman primates, uricase catalyzes the conversion of uric acid to allantoin. However, in humans, uricase deficiency leads to decreased uric acid excretion and –increased serum accumulation. Thus, when serum uric acid content exceeds certain levels (149–416 µmol/L in males and 89–257 µmol/L in females) and unable to be excreted, it leads to chronic hyperuricemia (Rosas et al., 2020). Eventually, this will lead to gout as a result of the deposition of urate microcrystals in and around joints (Pascart & Liote, 2019). Purine content varies widely across different foods. For example, the purine content in vegetable foods has been reported to be extremely low, while foods such as seafood, beer, and organ meats are relatively high in purines. People with impaired purine metabolism, such as those with hyperuricemia or gout, are advised to limit the consumption of such high-purine foods (Khanna et al., 2012). Observed gender differences in serum uric acid suggest that female hormones, including estrogen and progesterone, may decrease uric acid levels (Akasaka et al., 2014; Furuhashi, 2020). Recent studies have shown that serum uric acid may have some proinflammatory, prooxidative, and vasoconstrictive effects which may contribute to cardiometabolic diseases (Borghi et al., 2020). Therefore, correctly elucidating the purine content of different foods is essential.

FIGURE 1. The structural formula of the adenine, guanine, xanthine, and hypoxanthine

FIGURE 2. The absorption and excretion of uric acid

FIGURE 3. Purine metabolic pathway. Abbreviations: ADA: Adenosine deaminase; XOD: Xanthine oxidase; 5′-NT: Nucleotidase; Purine nucleoside phosphorylase

Due to the importance of purine content in foods to human health, this review sorts to summarized the common pretreatment and quantification methods that have been used in recent years for the determination of purines in different foods. The resulting guanine, adenine, xanthine, and hypoxanthine contents in the different foods were also summarized.

PRETREATMENT FOR PURINES EXTRACTION

Purines in food mainly exist in the form of nucleic acid, nucleotide, nucleoside, and free purine bases. To measure purine content, bound purines such as in nucleic acid needs to be dissociated into the four free purine bases before further determination. Several methods have been reported in the literature as pretreatment to allow for the dissociation of bound purines.

Acid hydrolysis

Acid hydrolysis is the most common for purine extraction in foods. It is further divided into different hydrolysis methods according to the type of acid used. The commonly used acids include perchloric acid, trifluoroacetic acid, trichloroacetic acid (TCA), formic acid, sulfuric acid, phosphoric acid, hydrochloric acid, acetic acid, or mixed acids. Among these, perchloric acid has a better hydrolysis effect on purines, but the acid concentration and hydrolysis time have been different across different studies. Some studies involving the use of acid hydrolysis are listed in Table 1.

TABLE 1 Acid hydrolysis methods for purine extraction in food

Acid hydrolysis	Dosage	Temperature (℃)	Time (min)	Sample	Reference
Perchloric acid	10 mL, 70–72%	100	60	Beer	Li et al. (2018)
Perchloric acid	10 mL, 80%	80	55	Marine fish	Li et al. (2019)
Perchloric acid	2 mL, 6.0%	100	60	Pork, beef	Rong et al. (2015)
Perchloric acid	70%	100	60	Sweet potato and beef	Inazawa et al. (2014)
Sulfuric acid	2 mL, 3 mol/L	100	5	Beer	Li et al. (2018)
Sulfuric acid	1.0 mL, 98%	100	120	Calf thymus	Liu et al. (2008)
Methanol–hydrochloric acid	44 mL, 40:4, v/v	85	90	Tea	Nian et al. (2019)
Hydrochloric acid	10 mL	98	60	Beer	Yuru (2008)
Trifluoroacetic acid–formic acid–water	11 mL, 5:5:1, v/v	90	12	Marine products	Qu et al. (2017)
Trifluoroacetic acid–formic acid	10 mL, 1:1	100	60	Beer	Yuru (2008)

Perchloric acid hydrolysis

Perchloric acid is one of the most commonly used acids in acid hydrolysis, primarily due to its better nucleic acid hydrolysis ability. Purines in natural and cultured Cordyceps were hydrolyzed (Fan et al., 2007) using different acids including perchloric acid, sulfuric acid, formic acid, phosphoric acid, and hydrochloric acid. Perchloric acid was found to better hydrolyze the purines. The best hydrolysis condition was in eight folds pure commercial perchloric acid (concentration of commercial perchloric acid was 70%) for 1 h at 95–100°C. Cauliflower homogenate was hydrolyzed to extract purines using 70% perchloric acid (Yamaoka et al., 2010). The results showed that purine content in the heated cauliflower was slightly higher than that in the raw cauliflower. In another study, pork and beef were hydrolyzed for purine extraction with 6% perchloric acid for 60 min over 100°C water bath (Rong et al., 2015). Hairtail, eel, and grass carp were hydrolyzed to extract purines with 10% perchloric acid (Guo et al., 2020). The mixture was heated at 100°C for 60 min. Finally, nucleic acids, nucleosides, and nucleotides were mostly hydrolyzed into corresponding purine bases. Some studies have mentioned that the hydrolysis of nucleoside bases with a high concentration of perchloric acid may lead to the degradation of the bases. However, hydrolysis with a high concentration of perchloric acid shows that degradation is not dominant. Degradation, however, becomes dominant upon complete hydrolysis. Therefore, attention should be paid to the perchloric acid concentration and the endpoint of the hydrolysis reaction in the pretreatment of the sample in order to optimize the process (Fan et al., 2007).

Sulfuric acid hydrolysis

Sulfuric acid hydrolysis is an improved pretreatment with characteristics of mild hydrolysis, short hydrolysis time, and no precipitation after neutralization. However, with a longer hydrolysis time (60 min), oxidation of purine bases occurs. Conversely, with a short hydrolysis time (5 min), the amount of purines extracted is greatly decreased. Therefore, the selection of experimental conditions should be given a great consideration when purine is extracted by sulfuric acid hydrolysis. In a study, bound purines including xanthine, hypoxanthine, guanine, and adenine in different brands of beer were totally hydrolyzed with 3 mol/L sulfuric acid over 5 min treatment time (Li et al., 2018). When the acid concentration was reduced to below 3 mol/L, the purines were only partially hydrolyzed. On the other hand, increasing the acid concentration beyond 3 mol/L led to increased purine oxidation and reduced detectable purine content. However, hydrolysis of calf thymus DNA using 98% (18 mol/L) sulfuric acid at 100°C for 2 h (Liu et al., 2008) resulted in a good purine-bases separation. Under the same conditions, guanine, adenine, xanthine, and hypoxanthine content in beer hydrolyzed by 3 mol/L sulfuric acid was observed to be 66.54, 40.82, 39.30, and 3.76 mg/L, respectively. However, when beer was hydrolyzed with 70% perchloric guanine, adenine, xanthine, and hypoxanthine yield was observed to be 50.47, 23.20, 35.73, and 2.89 mg/L, respectively; indicating a relatively lower yield compared to sulfuric acid hydrolysis (Li et al., 2018). Therefore, due to its high extraction rate, sulfuric acid hydrolysis may be a better pretreatment option for beers. These also suggest that the optimum sulfuric acid concentration range for purine hydrolysis is lower and narrower for liquid foods compared to nonliquid products.

Trifluoroacetic acid/formic acid hydrolysis

Trifluoroacetic acid/formic acid hydrolysis is also a frequently used acid hydrolysis method. Compared to other acid hydrolysis methods, this method is characterized by a lower loss of hydrolyzed purines and a better hydrolysis effect. In one study, purines in beer were hydrolyzed by trifluoroacetic acid/formic acid (trifluoroacetic acid: formic acid, 1:1, v/v) and compared to hydrolysis by other acids. Through high-performance liquid chromatography (HPLC) analysis, it was found that the mixed acid (trifluoroacetic acid/formic acid) resulted in a better separation of the four purine bases compared to perchloric acid, phosphoric acid, acetic acid, and hydrochloric acid hydrolysis (Yuru, 2008). In another study, purines in seafood such as Lateolabrax japonicus, Scophthalmus maximus, and Scomberomorus niphonius, etc. were hydrolyzed with trifluoroacetic/formic acids (trifluoroacetic acid: formic acid : water, 5:5:1, v/v/v) over 90°C water bath for 12 min to extract purines (Qu et al., 2017). The HPLC analysis showed a purine recovery of 91.5–105.0%.

Supercritical fluid extraction

Supercritical fluid extraction (SFE) is a newly developed technology commonly used for the extraction of purine alkaloids in plant leaves for its good separation effect and environment friendliness. The purine alkaloids in Yerba Mate Leaves were extracted with supercritical CO₂ at 400 bar and 40°C (Teofilovic et al., 2018). In another study, the caffeine in wet ground guarana seeds and mate tea leaves were extracted with supercritical CO₂ at 400 bar and 70°C (Marleny et al., 2002). The purine alkaloids in Ilex paraguariensis leaves were extracted with supercritical CO₂ at 255 bar and 70°C (Saldana et al., 1999). Compared to other separation techniques, SFE is capable of extracting purines at moderate temperatures and the possibility to obtain pure purines with no traces of solvent.

Solid-phase extraction

Solid-phase extraction (SPE) has good selectivity, so it is often used for liquid sample purification and elution to improve sample purity. The purine alkaloids in Yerba Mate Leaves were separated from other substances by SPE column and eluted with dichloromethane to obtain the purine alkaloids (Teofilovic et al., 2018). Purines in beer, wine, sake, and liquor were extracted using a strong cation-exchange SPE column (Kakigi et al., 2014). Furthermore, 23 purine and pyrimidine compounds from rat serum were extracted by SPE. Thus, this method is suitable for the extraction of purines in large-scale metabolomic studies of liquid-based foods due to its high selectivity and specificity (Liu et al., 2019).

Trichloroacetic acid

TCA is usually used to separate protein-bound purines from protein as a purification measure. Hitherto sodium tungstate, perchloric acid, zinc sulfate, and TCA were commonly used for the separation. Blood samples were deproteinized with 10% TCA (Czauderna & Kowalczyk, 1997). In addition, nucleotides in leaves could be directly extracted by the TCA (Hajirezaei et al., 2003; Penghui et al., 2019). However, no comparison between the TCA and other acids was mentioned so that the comparative effect of TCA extraction was unknown. This method, however, allowed for the measurement of purine contents using reversed-phased and ion-exchange high-pressure liquid chromatography.

Solvent extraction

Solvent extraction is one of the food-purine extraction methods. Usually, methanol, ethanol, and water are used as extraction solvents. However, the effect of extraction is affected by the proportion of the solvent components. Eight purines and their metabolites of a typical marine bivalve mollusk Mactra veneriformis were extracted with different concentrations of methanol–water solution and ethanol–water solution (Ji et al., 2013). After HPLC analysis and comparison, it was found that the methanol–water solution (1:1) had a better extraction effect.

Three-phase hollow fiber liquid-phase microextraction

The advantages of the three-phase hollow fiber liquid-phase microextraction (HF-LPME) are simplicity, low-cost, a requirement for only small volumes of solvents, and excellent sample cleanup. Compared with the two-phase HF-LPME, the three-phase extraction produces purer purines and is particularly suitable for acidic and alkaline compounds. Complex sample matrices interfered with the determination of purines. To overcome these drawbacks, the three-phase HF-LPME is used for the enrichment of purines and to separate purines from other impurities. As a new extraction technology, liquid phase micro-extraction has higher sensitivity than the traditional extraction methods and can be used for the extraction of trace targets. In addition, it is suitable for a wide range of solvents at a relatively lower solvent volume. The application of this method allowed for the extraction of Alysicarpus vaginalis (L.) DC. powder purines (Liu et al., 2014). By comparing the effects of extraction solvent, pH of donor and acceptor phases, extraction time, stirring speed, and salt addition to the donor phase on the extraction effect, the optimum extraction conditions were determined as follows: n-octanol as organic solvent, pH of donor and acceptor phases as 10.0 and 3.5, respectively, extraction time of 40 min, the stirring speed of 800 rpm, and donor phase addition of salt of 10% (w/w). In addition, microwave-assisted extraction, ultrasonic-assisted extraction, thermal extraction, and other methods have also been used for extraction. However, acid hydrolysis has been the most commonly used method for the pretreatment of purines.

DETERMINATION OF PURINES Ion chromatography

With many dissociating groups, purine can be detected by ion chromatography under appropriate conditions. Due to the limitations of early ion chromatography columns and efficiency issues of ion chromatography analysis, it is now less frequently used since the development of liquid chromatography. However, it has also been reported by some literature that it had a simple operation and high selection in detection. The principle of ion chromatography is illustrated in Figure 4. The negatively charged stationary phase adsorbs the positively charged molecules in the mobile phase, and the negatively charged molecules then flow along. Four purines and pyrimidine bases (cytosine, 5-methylcytosine, adenine, and N⁶-methyladenine) from calf thymus were simultaneously quantified using ion chromatography and conductivity detection in an acidic medium. The limit of detection (LOD) of cytosine, 5-methylcytosine, adenine, and N⁶-methyladenine was 0.05, 0.08, 0.07, and 0.07 mg/L, respectively. It was found that the recovery of adenine in calf thymus DNA was more than 98% (Liu et al., 2008).

FIGURE 4. The schematic diagram of ion chromatography

Capillary electrophoresis

Capillary electrophoresis (CE) is a relatively widely used electrophoresis method in food purine detection. The principle of the electrophoresis is shown in Figure 5. Charged molecules move under the action of an electric field. Molecules with high molecular weight get intercepted by the gel's network structure and remain in the upper layer, while molecules with low molecular weight can penetrate the network structure and remain in the lower layer. Thus, electrophoretic bands are formed. In one typical study, eight kinds of catechins and three kinds of xanthine derivatives (caffeine, theophylline, and theobromine) in tea were simultaneously determined using CE (Bonoli et al., 2003). The catechins and xanthine derivatives were separated successfully in a phosphate-borate-SDS buffer system within 4.5 min, and the LOD ranged from 0.0051 to 0.011 µg/mL. This method is simple, rapid (the whole analysis time is about 12 min), reliable, sensitive, and does not require any sample pretreatment.

FIGURE 5. The schematic diagram of electrophoresis

Fast-scan cyclic voltammetry

Purine is a vital extracellular signal molecule in the brain, responsible for the regulation of nerve transmission and protection. Extracellular purine signaling molecules have two signal patterns in the brain: slow (in the order of minutes to hours) and fast (milliseconds to seconds). In order to detect fast-mode signal molecules, enzyme-coated electrodes with amperometric detection, aptamer-based sensors, and fast-scan cyclic voltammetry (FSCV) detection at carbon-fiber microelectrodes are most often used (Figure 6). Specifically, due to its subsecond temporal resolution and excellent spatial resolution, FSCV detection at carbon-fiber microelectrodes has been widely used for fast purine detection. However, due to the limited interaction between electrode surfaces, it is still a challenge for the detection of purine nucleotides, GTP, and ATP on bare carbon-fiber microelectrodes (CFME). The CFME is often treated with N₂ and O₂ plasma to improve the FSCV for fast purine detection, due to the significant effect on the detection of cationic and anionic purines in FSCV with the function and topology of carbon-fiber surface manipulated by N₂ and O₂ plasma. The sensitivity and adsorption strength on electrodes of purines are improved due to O₂ plasma. The detection of ATP might be improved due to the supply of amine groups by N₂ plasma on the electrodes surface (Li & Ross, 2020).

FIGURE 6. The schematic diagram of FSCV detection. The extracellular adenosine, guanosine, ATP, and GTP can be detected by plasma etcher-treated electrode connected with FSCV for subsecond rapid detection

Enzyme-assisted electrochemical detection

The fast and accurate detection of intracellular purines is very vital to understanding the physiological processes related to purine metabolism. However, the overlapping signals of guanine or xanthine and adenine or hypoxanthine hinder the accurate detection of individual purine alterations in cells. A novel enzyme-assisted electrochemical detection system for intracellular purification using multiwall carbon nanotubes-ionic liquid (MWCNTs-IL) modified glassy carbon electrode has been constructed by the addition of xanthine oxidase (Cui et al., 2015). In the detection process, hypoxanthine or xanthine is catalyzed by xanthine oxidase to uric acid. Using this enzyme-assisted electrochemical detection system, the individual concentrations of guanine, xanthine, adenine, and hypoxanthine in human breast cancer (MCF-7) cells were detected simultaneously without any pretreatment. Thus, this is a simple but rapid method to obtain more information about purine metabolism in cells.

Combined surface enhanced Raman spectroscopy diazotization and xanthine oxidase method

Diazotization is the reaction of guanine and adenine with nitrous acid at low temperatures to produce diazonium salt. The diazo group is then substituted by –H, –OH, –X, –CN, –NO, and other ion groups to yield xanthine and hypoxanthine. Xanthine oxidase could then catalyze the conversion of hypoxanthine to xanthine and then uric acid, and/or catalyze xanthine to uric acid directly. Therefore, total purine can be completely converted into uric acid by the combined diazotization and xanthine oxidase method. The uric acid concentration can then be determined by surface-enhanced Raman spectroscopy (SERS; Figure 7). Thus, total purine can be determined indirectly. This method was used to determine total purine contents in fish (Guo et al., 2020). Besides, the best diazotization and xanthine oxidase conditions were optimized for efficient performance. The optimal temperature and time of the diazotization reaction were 60°C for 40 min, respectively, and the enzyme reaction was fixed at 30°C for 40 min. The calibration curve established at the characteristic Raman peak at 631 cm⁻¹ had a good linear relationship (R² = 0.9864). The recovery rate was between 101.7% and 105.0%. The LOD was determined as 0.01 mmol/L. Therefore, the total purine content in fish can be accurately quantified with highly sensitive using this method.

FIGURE 7. The schematic diagram of SERS diazotization and xanthine oxidase

High-performance liquid chromatography

Purines in food have often been measured by HPLC. It is suitable for a wide range of foods including plants, fungus, and animal origins. However, the appropriate pretreatment for samples is required. In addition, quantification might be affected by the chromatographic column, mobile phase concentration, pH, flow rate, column temperature, and detection wavelength. Thus, it is crucial to choose the best chromatography condition. In a study, purines in marine fish and aquatic products were estimated using HPLC with an Agilent Eclipse XDB-C18 chromatography column (4.6 mm × 250.0 mm × 5.0 mm) (Li et al., 2019). The LOD of adenine, guanine, hypoxanthine and xanthine was 0.0774, 0.0178, 0.0118, and 0.0555 mg/L, respectively. It was found that the content of purine in the viscera of marine fish was significantly higher than that in muscle while adenine, guanine, and hypoxanthine were enriched in the viscera. In addition, purines in different parts of pork and beef were estimated using HPLC with a Waters Atlantis T3 chromatography column (Rong et al., 2015). It was found that hypoxanthine and adenine content in pig buttocks and beef loin were higher than those in the other parts (p < 0.05). Fukuuchi and co-workers established a more sensitive method to detect purine content in samples with lower concentrations (Fukuuchi et al., 2013). By comparing the separation effects of purines with different proportions of mobile phase solvent, 150 mmol/L sodium dihydrogen phosphate buffer (H₃PO₄:NaH₂PO₄ = 20:100, v/v) was used as the mobile phase. Besides, the LOD for adenine, guanine, hypoxanthine and xanthine was 0.0038, 0.0038, 0.0075, and 0.0075 mg/L, respectively. Therefore, the method by Fukuuchi et al. seem to be more sensitive. In addition, traditional pretreatment processes are easily affected by errors caused by small changes in the number of liquids in the degassing or neutralization steps. Thus, in order to reduce the dilution rate and improve accuracy, the pretreatment method is simplified. Due to the rich content of compounds in many foods and beverages, it is difficult to accurately identify and quantify purine contents. A sensitive and simplified HPLC method, the enzymatic peak-shift method, has been used to determine purine content by comparing the peaks of enzyme-treated samples with those of untreated samples. The HPLC system was equipped with a Shodex AsahipakGS-320HQ column (7.6 mm ID × 300 mm, 6.0 µm). In addition, it is suitable for the determination of purines in foods, cells, and biomaterials without interference from other compounds (Fukuuchi et al., 2013).

Reversed-phase high-performance liquid chromatography

All methods introduced in 3.6 are normal-phase HPLC, which consists of polar stationary and weak polar mobile phase, commonly used to separate compounds with moderate and strong polarity. However, reversed-phase high-performance liquid chromatography (RP-HPLC) is the most widely used modern liquid chromatography, accounting for about 80% of all HPLC applications. It is composed of a nonpolar stationary phase and polar mobile phase, which is often used to separate nonpolar, polar, or ionic compounds. Thus, the application range of RP-HPLC is wider than that of the normal-phase HPLC. Purine bases in the gonads of sea urchins with different storage times were determined by RP-HPLC with a Spherisorb ODS 2 C-18 chromatography column (PinEiro-Sotelo et al., 2002). The LOD for adenine, guanine, hypoxanthine and xanthine was 0.076, 0.04, 0.06, and 0.068 mg/L, respectively. The results showed that the concentration of adenine, guanine, and xanthine decreased with increasing storage time whiles hypoxanthine increased. Thus, it might be inferred that purine contents might be related to the freshness of the food. Furthermore, purines in fish oil supplements were estimated using RP-HPLC with Phenomenex Luna Silica analytical column (Roy et al., 2013). The LOD of adenine, guanine, hypoxanthine, and xanthine was 0.01 mg/L. Thus RP-HPLC is a simple, efficient, and reproducible method for the analysis of purines in fish oil and other foods.

HPLC-electrospray ionization/MS

Most foods including the active ingredients in traditional Chinese medicine contain many very complex compounds, as high as in the hundreds. For a complex compound system, similar retention time and UV spectra of purines and pyrimidines by HPLC or RP-HPLC result in a reduced separation sensitivity. On the contrary, due to the high sensitivity and excellent detection of HPLC-electrospray ionization (ESI)/MS, it enables the detection of complex compound systems with sensitivity. Ten nucleosides and bases in bulbus Fritillariae cirrhosae were determined using HPLC-ESI/MS with an Agilent Zorbax SB-AQ C18 chromatography column (Duan et al., 2011) and the obtained LOD ranged from 0.04 to 0.20 mg/L. The LOD of purines was lower than the others. Importantly, 2-deoxyadenosine and hypoxanthine were isolated for the first time. Thus, providing a solid basis for the pharmacological research into F. cirrhosae. HPLC methods usually produce very low LOD in purine detection, as an indication of high sensitivity. Therefore, HPLC has become an attractive method for the detection of purines in recent times. Different chromatography conditions including chromatography column type, flow rate, column temperature, detection wavelength, mobile phase composition, mobile phase concentration, and mobile phase pH will be required for different samples. Some frequently used HPLC conditions for the determination of purine content in foods are shown in Table 2.

TABLE 2 HPLC conditions in the determination of purine content in food

Chromatography column	Flow rate (mL/min)	Column temperature (°C)	Detection wavelength (nm)	Mobile phase	Sample	Reference
Zorbax Eclipse XDB-C8	0.8	25	273	Water (pH 8) + acetonitrile (v/v = 4:1)	Yerba mate folium	Teofilovic et al. (2018)
Thermo BDS Hypersil C18	1	25	254	20 mmol/L sodium phosphate buffer, pH 4.21	Beer	Li et al. (2018)
Agilent Eclipse XDB-C18	0.8	28	254	Water–methanol–glacial acetic acid-20% tetrabutylammonium hydroxide (v/v = 879/100/15/6, pH 3.44)	Marine fish	Li et al. (2019)
Shodex Asahi Pak GS-320HQ	0.6	35	260	150 mmol/L H3PO4/NaH2PO4 (v/v = 40/100 pH 2.4; 35/100 pH 2.5; 30/100 pH 2.6; 25/100 pH 2.7; 20/100 pH 2.8; 15/100 pH 2.9)	Beer and beer-like alcoholic beverages	Fukuuchi et al. (2013)
CAPCELL PAK UG120	1.0	40	270	A: water/triethylamine/phosphoric acid = 950/10/5 (v/v), pH 3.8 B: water/acetonitrile = 90/10 (v/v)	Marine Foods	Ishimaru et al. (2015)
RP column KinetexTM C18	0.5	25	254	A: 48 mmol/L KH2PO4 and 2 mmol/L K2HPO4 with methanol (97:3 = v/v) pH 4.0–6.5 B: 50 mmol/L (or 0.10 mol/L and 0.20 mol/L) acetate buffer, pH 4.0 and methanol (97:3 = v/v)	—	Markelj et al. (2016)
Atlantis dC18	1.5	27	270	0.1% formic acid, pH 3.75 with ammonia	Tea	Oduro-Mensah et al. (2018) and Zhou et al. (2020a)
Agilent Poroshell 120 EC-C18	0.6/0.8/1.0	30/35/40	280	A: 0-0.2% ortho-phosphoric acid B: 80% acetonitrile, 80% methanol 40% acetonitrile or 40% MeOH at three column temperatures	Tea	Nian et al. (2019)
BEH shield RP18	0.125	35	254	A: 0.1% formic acid B: acetonitrile	Pork meat	Clariana et al. (2010)
Waters Atlantis T3	1	30	254	99% 10.0 mmol/L ammonium formate buffer, pH 3.6, 1% methanol	Beef and pork	Rong et al. (2015)
Zorbax Eclipse Plus C18	0.3	25	270	A: 0.5% acetic acid B: methanol	Theobroma cacao	Cádiz-Gurrea et al. (2014)
HSS T3	0.3	—	260	A: 1.25 mmol/L dehydroascorbic acid in 10 mmol/L formic-acid-ammonium-formate buffer, pH5.0 B: 1.25 mmol/L dehydroascorbic acid in acetonitrile	Sweet potato, raw beef, and cooked beef	Inazawa et al. (2014)
Waters Atlantis dC18	1.0	30	254	0.02 mol/L potassium phosphate monobasic solution, pH 3.6	Seafood	Qu et al. (2017)
Agilent C18	1.5	27	270	A: 100% acetonitrile B: 0.1% ammonium formate (adjusted to pH 3.75 with ammonia)	—	Oduro-Mensah et al. (2018) and Zhou et al. (2020b)
Nova-Pak C18	0.5	—	272	methanol–water–acetic acid (7.5:92:0.5 v/v/v)	Fish	Sen and Sarkar (2020)
C18 Synergi MAX-RP	0.8	30	270 for XA 250 for others	A: 50 mmol/L potassium phosphate buffer, pH 4.0 B: 20% methanol C: 50% methanol	Dry-cured ham	Hernandez-Cazares et al. (2011)
Zorbax Eclipse XDB-C18	1	30	254	A: 5 mmol/L PIC (tetrabutylammonium hydrogen sulfate/phosphoric acid in water) in 100 mmol/L phosphate buffer, pH6 B: 75% A and 25% methanol	Pork	(Mora et al. (2010)
Prodigy ODS-3	—	—	245	A: methanol/water (60:40) B: 0.02 mol/L KH2PO4, pH 5.5	Chicken muscle	Aliani and Farmer (2005)
Wakopak Navi C22-5 reverse-phase column	1	35	250	50 mmol/L sodium di-hydrogen phosphate, pH 4.0	Fish	Kuda et al. (2008)
SynergiTM Hydro-RP LC	0.2	30	—	0.05 mol/L acetic acid buffer: adjusting to pH 4.0 with ammonium solution and readjusting to pH 2.8 with formic acid A: 10% methanol, 0.05 mmol/L acetic acid buffer B: 50% methanol, 0.05 mmol/L acetic acid buffer	Bovine blood plasma	Stentoft et al. (2014)
BioBasic-C18	0.5	30	254	A: methanol B: water	Mactra veneriformis	Ji et al. (2013)
YMC C18	1.0	30	254	A: water B: methanol	Alysicarpus vaginalis (L.) DC.	Liu et al. (2014)

TOTAL PURINE CONTENT IN VARIOUS FOODS

According to the level of purine content, foods can be divided into high and low purine foods. High purine food isis food that can rapidly cause an increase in purine content in the blood after ingestion. Different high purine foods contain different kinds of purines. In this review, foods were divided into plant foods, fungus and algae foods, animal foods, and aquatic products. The total purine content was in the order plant foods < fungus and algae foods < animal foods < aquatic products.

Purine content in plant foods

In this paper, plant foods were categorized into cereal and cereal products, nuts, legume and legume products, vegetable and vegetable products, and fruits (Kaneko et al., 2014, 2020; Shengzhong et al., 2012). The analysis (Figure 8) showed that purine content in legumes was the highest, with an average of 59.44 mg/100 g (ranged from 7.4 to 293.1 mg/100 g), followed by nuts with an average of 51.50 mg/100 g (ranged from 19.6 to 71.3 mg/100 g). Cereals and cereal products, vegetables and vegetable products, and fruits are generally lower in purine content, not exceeding 70 mg/100 g, except parsley and wheat malt which reached 288.9 and 135.4 mg/100 g, respectively. In addition, the content of adenine and guanine in other plant foods including legumes generally account for more than 60% of the total purine content. Besides a few plant foods have higher hypoxanthine and xanthine contents. For example, hypoxanthine and xanthine contents in wheat malt account for more than 40% of the total purine content.

FIGURE 8. Heat map of purine content in plant foods. (a) Purine content in legume and legume products. (b) Purine content in cereal and cereal products. (c) Purine content in fruits. (d) Purine content in vegetable and vegetable products. (e) Purine content in nuts. Abbreviations: A, adenine; G, guanine; H, hypoxanthine; T, total purine; X, xanthine

Purine content in fungus and algae foods

Total purines contents in 25 fungi and algae foods (Kaneko et al., 2014) were reviewed in this paper (Figure 9). White Aragekikurage had the lowest total purine content (6.9 mg/100 g). Those with total purine content of more than 100 mg/100 g were Hijiki (dried), Hiratake, Jew's-ear (dried), Shiitake (for broth, dried), Wakame (dried), Lentinula edodes, and Nori (dried). The total purine in Nori (dried), 591.70 mg/100 g, was the highest. Hypoxanthine and xanthine contents in almost all the bacteria and algae foods were lower than adenine and guanine, accounting for about 10% of total purines.

FIGURE 9. Heat map of purine content in fungus and algae. Abbreviations: A, adenine; G, guanine; H, hypoxanthine; T, total purine; X, xanthine;

Purine content in animal foods

Here, animal foods were categorized into poultry, livestock, processed meat products, and dairy and eggs (Hong-Zhi et al., 2012; Kaneko et al., 2014, 2020; Xin-Yan et al., 2008; Yun et al., 2008) (Figure 10). We found great variation in purine contents in different kinds of animal foods. Processed meat, dairy, and eggs had lower purine contents, with an average of 75.71 and 6.58 mg/100 g, respectively. The total purine contents in livestock and poultry were generally higher than 100 mg/100 g. In the liver, kidney, and heart, purine contents were generally higher, with poultry liver being higher than that of livestock. The purine contents of pig liver, bovine liver, chicken liver, and duck liver were 284.80, 219.80, 312.20, and 317.60 mg/100 g, respectively. The proportion of hypoxanthine and xanthine in processed meat products was relatively high, while that of livestock and poultry was relatively low. In the process of cooking, the purine in the meat will be dissolved in the soup, which reduces the purine content in the meat and increases the purine content in the soup.

FIGURE 10. Heat map of purine content in animal foods. (a) Purine content in poultry. (b) Purine content in processed meat products. (c) Purine content in dairy and eggs. (d) Purine content in livestock. Abbreviations: A, adenine; G, guanine; H, hypoxanthine; T, total purine; X, xanthine

Purine content in aquatic products

Aquatic products were divided into fresh, fermented, dried, and processed products (Figure 11). The purine content of aquatic products was generally above 100 mg/100 g, and as high as over 1000 mg/100 g (Kaneko et al., 2014, 2020; Lou, 2010; PinEiro-Sotelo et al., 2002; Takako et al., 1981). The purine contents of shrimps and shellfish were higher than that of fish. Besides, hypoxanthine, adenine, and guanine contents accounted for a higher proportion of the total purine content. This may account for the rapid increase in bodily uric acid upon the intake of these kinds of foods. Dried fish had higher purine content compared to half-dried fish. However, it is a vital food source for humans because it is rich in protein, minerals, and other nutrients. Some related studies have found that the purine content of surimi can be greatly reduced by repeated washing. But cooking and barbecue will increase the content of adenine and guanine (Lou et al., 2005). Thus, patients with hyperuricemia can eat surimi washed repeatedly to satisfy the needs of nutrition.

FIGURE 11. Heat map of purine content in aquatic products. (a) Purine content in the fresh. (b) Purine content in processed products. (c) Purine content in the dried. (d) Purine content in the fermented. Abbreviations: A, adenine; G, guanine; H, hypoxanthine; T, total purine; X, xanthine

Purine content in condiments and supplements

Some common condiments and supplements contain purines (Kaneko et al., 2014, 2020). However, the kinds and contents differ greatly (Figure 12). Because of the fresh-keeping effect of fresh soup powder, the purine content of fresh-keeping agents such as fresh soup powder reached 684.8 mg/100 g. But the purine content of spirulina, brewer's yeast, Chlorella, and DNA/RNA supplements was 1076.80, 2995.70, 3182.70, and 21,493.60 mg/100 g, respectively. These kinds of food additives have naturally high purine or nucleic acid content. Thus, patients with hyperuricemia should consume these with caution.

FIGURE 12. Heat map of purine content in condiments and supplements. Abbreviations: A, adenine; G, guanine; H, hypoxanthine; T, total purine; X, xanthine;

Suggestions for patients with hyperuricemia or gout

As excessive intake of purine in food could lead to hyperuricemia and gout, it is necessary to take some measures to detect purine substances in food in order to provide corresponding dietary suggestions for patients with hyperuricemia or gout. It is advisable to consume more vegetables, fruits, and grains with low purine content. Surimi that has been washed repeatedly, pork, chicken, and so on could be good choices as sources of protein. However, meat must be removed from the soups after cooking. In this way, a balanced diet can be achieved.

CONCLUSION AND FUTURE PERSPECTIVES

Hyperuricemia is considered one of the most important metabolic disorders along with hyperlipidemia, hypertension, and hyperglycemia, attracting increasing attention. Because hyperuricemia is a metabolic syndrome caused by a high purine diet, it is very important to control the intake of dietary purine reasonably. In this paper, the common methods for the detection of purine in food were reviewed, including pretreatment, ion chromatography, electrophoresis, electrochemical detection, and HPLC. Different methods have different applicable conditions and characteristics; therefore, the best method must be selected according to different detection purposes, food material, and experimental conditions. With the increasing technological advancement, methods for the detection of food purine are equally gradually advancing. For example, pretreatment is more ideal, separation is more precise and detection is more accurate. We have summarized the purine content in common foods not only to provide dietary advice for special populations but also to provide a scientific basis for future research on hyperuricemia and gout.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the Natural Science Foundation of Guangzhou Province of China (2019A1515012230), the National Natural Science Foundation of China (No. 32072207), and the Fundamental Research Funds for the Central Universities (2019KZ01).

CONFLICT OF INTERESTS

The authors declare that they have no conflict of interests.