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Uric acid shown to contribute to increased oxidative stress level independent of xanthine oxidoreductase activity in MedCity21 health examination registry

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• Published: 01 April 2021

Uric acid shown to contribute to increased oxidative stress level independent of xanthine oxidoreductase activity in MedCity21 health examination registry

• Masafumi Kurajoh, 
• Shinya Fukumoto, 
• Shio Yoshida, 
•  

Scientific Reports volume 11, Article number: 7378 (2021) Cite this article

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Abstract

Uric acid has both antioxidant and pro-oxidant properties in vitro by scavenging and production of reactive oxygen species (ROS). This cross-sectional study examined whether uric acid possesses effects on oxidative stress under physiological conditions independent of xanthine oxidoreductase (XOR), which is involved in uric acid and ROS production. Serum uric acid level was measured, while plasma XOR activity was determined using our high-sensitive assay in 192 participants (91 males, 101 females) who underwent health examinations and were not taking an antihyperuricemic agent. For antioxidant potential and oxidative stress level, biological antioxidant potential (BAP) and derivative of reactive oxygen metabolites (d-ROMs) in serum, respectively, were measured. Median uric acid level and plasma XOR activity were 5.6 mg/dL and 26.1 pmol/h/mL, respectively, and BAP and d-ROMs levels were 2112.8 μmol/L and 305.5 Carr U, respectively. Multivariable regression analyses revealed no significant association of serum uric acid level with BAP level, whereas serum uric acid level showed a significant association with d-ROMs level independent of plasma XOR activity (p = 0.045), which was prominent in females (p = 0.036; p for interaction = 0.148). Uric acid might contribute to increased oxidative stress independent of XOR activity by increasing ROS production, without affecting ROS scavenging, especially in females.

요약

요산은

vitro에서 활성산소(ROS)를 소거하고 생성함으로써

항산화 및 산화 촉진 특성을 모두 가지고 있습니다.

이 단면 연구는

요산과 ROS 생성에 관여하는 크산틴 산화 환원 효소(XOR)와 무관하게

생리적 조건에서 요산이 산화 스트레스에 영향을 미치는지 여부를 조사했습니다.

혈청 요산 수치를 측정하고,

건강 검진을 받고

항고요산제를 복용하지 않는 192명의 참가자(남성 91명, 여성 101명)를 대상으로

고감도 분석을 통해 혈장 XOR 활성을 측정했습니다.

xanthine oxidoreductase (XOR)

항산화 잠재력과 산화 스트레스 수준을 측정하기 위해

혈청 내 생물학적 항산화 잠재력(BAP)과 활성 산소 대사 산물 유도체(d-ROMs)를 각각 측정했습니다.

요산 수치의 중앙값과 혈장 XOR 활성은 각각 5.6mg/dL과 26.1pmol/h/mL이었고,

BAP와 d-ROMs 수치는 각각 2112.8μmol/L과 305.5Carr U였습니다.

다변량 회귀 분석 결과,

혈청 요산 수치는 BAP 수치와 유의미한 상관관계가 없는 것으로 나타났습니다.

그러나 혈청 요산 수치는 혈장 XOR 활성(p = 0.045)과는 무관하게 d-ROM 수치와 유의미한 상관관계가 있는 것으로 나타났는데, 이는 여성에게서 두드러졌습니다(p = 0.036; 상호작용에 대한 p = 0.148).

특히 여성의 경우, 요산은 ROS 제거에 영향을 미치지 않으면서도 ROS 생성을 증가시켜 XOR 활동과 관계없이 산화 스트레스 증가에 기여할 수 있습니다.

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Introduction

Uric acid is produced by a xanthine oxidoreductase (XOR)-catalyzed reaction, and shown to have both antioxidant and pro-oxidant properties in vitro by scavenging and production of reactive oxygen species (ROS)1,2. However, its role in regulating oxidative stress under physiological conditions remains unclear, as such a reaction produces ROS as well as uric acid3,4.

Although several methods to determine circulating XOR activity have been reported, such as with use of an ultraviolet (UV) detector or liquid chromatography (LC)/UV5,6, there are difficulties with obtaining accurate measurements of circulating XOR activity in humans, as it is extremely lower than that in rodents7. We recently developed a highly sensitive test for human plasma XOR activity that utilizes assay results of stable isotope-labeled [13C2,15N2] xanthine obtained with LC/triple quadrupole mass spectrometry (TQMS)8,9. Using this method, associations of plasma XOR activity with serum uric acid level have been found in both cross-sectional and longitudinal studies10,11,12, suggesting that XOR activity in plasma reflects systemic XOR activity.

Some previous studies conducted in clinical settings have reported an association between uric acid and oxidative stress level13,14,15. However, to the best of our knowledge, no investigation of the association of uric acid with oxidative stress level adjustment for XOR activity in humans has been reported. To determine whether uric acid has an effect on oxidative stress level independent of XOR activity under physiological conditions, the associations of serum uric acid level and plasma XOR activity with levels of reactive biological antioxidant potential (BAP) and oxygen metabolites (d-ROMs), markers of antioxidant potential and oxidative stress, respectively16, were examined using our novel XOR activity assay in subjects who voluntarily underwent a health examination.

소개

요산은

크산틴 산화 환원 효소(XOR)에 의해 촉매 작용을 일으켜 생성되며,

체외에서 활성 산소 종(ROS)의 소거 및 생성을 통해 항산화 및 산화 촉진 특성을 모두 갖는 것으로 나타났습니다1,2.

그러나 이러한 반응은

요산뿐만 아니라 ROS도 생성하기 때문에

생리적 조건에서 산화 스트레스를 조절하는 요산의 역할은 아직 명확하지 않습니다3,4.

자외선(UV) 검출기 또는 액체 크로마토그래피(LC)/UV5,6를 사용하는 등 순환 XOR 활동을 측정하는 여러 가지 방법이 보고되었지만, 인간에서 순환 XOR 활동을 정확하게 측정하는 데 어려움이 있습니다. 설치류에 비해 매우 낮기 때문입니다7. 최근에 저희는 LC/트리플 쿼드러플러 질량 분석법(TQMS)8,9을 통해 얻은 안정 동위원소 표지 [13C2,15N2] 크산틴의 분석 결과를 활용하는 인간 혈장 XOR 활성에 대한 고감도 테스트를 개발했습니다. 이 방법을 사용하여, 혈장 XOR 활성과 혈청 요산 농도의 연관성이 단면 연구와 종단 연구에서 모두 발견되었으며10,11,12, 이는 혈장 내 XOR 활성이 전신 XOR 활성을 반영한다는 것을 시사합니다.

임상 환경에서 수행된 일부 이전 연구에서는 요산과 산화 스트레스 수준 간의 연관성을 보고했습니다13,14,15. 그러나 우리가 아는 한, 요산과 산화 스트레스 수준 간의 연관성에 대한 연구는 인간에서 XOR 활성을 조정하는 것으로 보고된 바가 없습니다. 생리학적 조건 하에서 요산이 XOR 활성과는 독립적으로 산화 스트레스 수준에 영향을 미치는지 확인하기 위해, 혈청 요산 수준과 혈장 XOR 활성을 각각 항산화 잠재력 지표인 반응성 생물학적 항산화 잠재력(BAP)과 산소 대사 산물(d-ROMs)과 연관지어 조사했습니다16. 이 연구는 자발적으로 건강 검진을 받은 피험자를 대상으로 새로운 XOR 활성 분석법을 사용하여 실시되었습니다.

Results

Clinical characteristics of subjects

Characteristics of the enrolled subjects are shown in Table 1. The median values for uric acid and plasma XOR activity were 5.6 mg/dL and 26.1 pmol/h/mL, respectively, while those for BAP and d-ROMs level were 2112.8 μmol/L and 305.5 Carr U, respectively.

Table 1 Clinical characteristics of subjects (n = 192).

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No association of serum uric acid level with BAP level

To examine whether uric acid level was independently associated with BAP level after adjustment for other confounding factors including plasma XOR activity, multivariable regression analyses were performed (Table 2). While Visceral fat area (VFA) and total cholesterol were significantly associated (p < 0.05), neither serum uric acid level nor plasma XOR activity showed a significant association with BAP level.

Table 2 Multivariable regression analysis of possible factors associated with BAP level.

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Association between uric acid and d-ROMs levels in serum is independent of plasma XOR activity

To examine whether serum uric acid level was independently associated with d-ROMs level after adjustment for other confounding factors including plasma XOR activity, multivariable regression analyses were performed (Table 3). Uric acid level, but not plasma XOR activity, was significantly associated with d-ROMs level (p = 0.045) (Figs. 1, 2), as were gender as well as VFA and high-sensitivity C-reactive protein (hs-CRP) (p < 0.05). On the other hand, no such association was seen with age, smoking habit, alcohol drinking habit, systolic blood pressure (SBP), total cholesterol, glycated hemoglobin (HbA1c), estimated glomerular filtration rate (eGFR), or homeostatic model assessment of insulin resistance (HOMA-IR).

Table 3 Multivariable regression analysis of possible factors associated with d-ROMs level.

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Figure 1

Serum uric acid and d-ROMs levels. The serum level of uric acid was significantly associated with d-ROMs level (p = 0.045) and the nonlinear effect of that on d-ROMs level was also significant (p = 0.171). Best fitted line and 95% CI results are shown by a solid line and gray band, respectively. Values for d-ROMs were adjusted according to the median values for age, gender, smoking habit, alcohol drinking habit, VFA, SBP, total cholesterol, HbA1c, eGFR, log HOMA-IR, log hs-CRP, and log plasma XOR activity. d-ROMs derivative of reactive oxygen metabolites, CI confidence interval, VFA visceral fat area, SBP systolic blood pressure, HbA1c glycated hemoglobin, eGFR estimated glomerular filtration rate, HOMA-IR homeostatic model assessment of insulin resistance, hs-CRP high-sensitivity C-reactive protein, XOR xanthine oxidoreductase.

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Figure 2

Plasma XOR activity and d-ROMs level. Plasma XOR activity was not significantly associated with d-ROMs level (p = 0.725). Best fitted line and 95% CI results are shown by a solid line and gray band, respectively. Values for d-ROMs were adjusted according to the median values for age, gender, smoking habit, alcohol drinking habit, VFA, SBP, total cholesterol, HbA1c, eGFR, log HOMA-IR, log hs-CRP, and uric acid level. XOR xanthine oxidoreductase, d-ROMs derivative of reactive oxygen metabolites, CI confidence interval, VFA visceral fat area, SBP systolic blood pressure, HbA1c glycated hemoglobin, eGFR estimated glomerular filtration rate, HOMA-IR homeostatic model assessment of insulin resistance, hs-CRP high-sensitivity C-reactive protein.

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Association of serum uric acid level with d-ROMs level stratified by gender

To further examine whether the association of uric acid level with d-ROMs level was affected by gender, interaction analyses were performed. The “gender * uric acid” interaction was significant (p = 0.148), while uric acid level was shown to be significantly associated with d-ROMs level in females (p = 0.036) but not males (p = 0.115) (Fig. 3), suggesting that gender has an effect on the relationship between uric acid and d-ROMs in serum. On the other hand, plasma XOR activity showed no significant association with d-ROMs level in either females (p = 0.350) or males (p = 0.394).

Figure 3

Serum uric acid and d-ROMs levels stratified by gender. The serum level of uric acid was significantly associated with d-ROMs level in females (p = 0.036) but not in males (p = 0.115). The nonlinear effect of serum uric acid level on d-ROMs level was significant (p = 0.066). Best fitted line and 95% CI results are shown by a solid line and dark gray band, respectively. Values for d-ROMs were adjusted according to the median values for age, smoking habit, alcohol drinking habit, VFA, SBP, total cholesterol, HbA1c, eGFR, log HOMA-IR, log hs-CRP, and log plasma XOR activity after stratification by gender. d-ROMs derivative of reactive oxygen metabolites, CI confidence interval, VFA visceral fat area, SBP systolic blood pressure, HbA1c glycated hemoglobin, eGFR estimated glomerular filtration rate, HOMA-IR homeostatic model assessment of insulin resistance, hs-CRP high-sensitivity C-reactive protein, XOR xanthine oxidoreductase.

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Discussion

This is the first known study to investigate the relationship of uric acid level and XOR activity with oxidative stress level in humans. In analyses of subjects who voluntarily underwent a health examination, our findings showed that the level of uric acid in serum had no significant association with BAP level independent of plasma XOR activity (Table 2). In contrast, serum uric acid level showed a significant association with d-ROMs level independent of plasma XOR activity (Table 3; Figs. 1, 2). Furthermore, the significant association of serum uric acid level with d-ROMs level was more prominent in females as compared to males (Fig. 3). Together, these results suggest that uric acid contributes to an increase in oxidative stress level by increasing ROS production under physiological conditions in a manner independent of XOR activity, especially in females. On the other hand, the antioxidant effects of uric acid under physiological conditions might be counterbalanced by ROS produced by an XOR-catalyzed reaction.

Oxidative stress is caused by a disturbance of the relationship between antioxidant activity and ROS production, and several markers have been proposed for eval‎uation of oxidative stress and antioxidant activity. Tests for BAP and d-ROMs, markers of antioxidant activity and oxidative stress, respectively, have been experimentally validated by results obtained with electron spin resonance, the gold standard of techniques actually available for free radical studies16,17. Furthermore, BAP testing has been shown to provide results comparable to other markers of antioxidant capacity, such as ferric-reducing ability and plasma antioxidant tests18,19, while a d-ROMs test was also demonstrated to provide results comparable to other markers of oxidative stress, such as FOX assay and 8-isoprostane20,21. Therefore, in the present study, we used BAP and d-ROMs tests to investigate the association of uric acid and XOR activity with antioxidant capacity and oxidative stress level.

In in vitro findings, uric acid has been shown to gain antioxidant properties by scavenging ROS, such as singlet oxygen, peroxyl radical, hydroxyl radical, and peroxynitrite1,22. Furthermore, serum uric acid levels were reported to be positively associated with markers of antioxidant potential in subjects who underwent a health examination14,15,23. However, those associations were not analyzed after adjustment for the activity of XOR, which produces ROS in addition to uric acid3,4. In the present results of multivariable regression analysis including plasma XOR activity as a covariate, uric acid was not significantly associated with BAP level (Table 2). Of interest, the antioxidant effects of uric acid in humans have been reported in examinations performed under non-physiological settings such as following exogeneous administration. For example, administration of uric acid or its precursor inosine improved endothelial function in patients with type 1 diabetes24, as well as clinical functional outcomes in patients with acute ischemic stroke25 or multiple sclerosis26. On the other hand, hyperuricemia was shown to be associated with endothelial dysfunction27,28 and used to predict poor clinical functional outcomes in patients with acute ischemic stroke29. Thus, the present results along with those in previous studies suggest that uric acid exerts antioxidant effects mainly under non-physiological conditions, such as exogeneous administration, while those effects are counterbalanced by ROS produced by an XOR-catalyzed reaction under physiological conditions.

In addition to antioxidant properties, uric acid is known to have pro-oxidant properties by generating ROS such as superoxide anions, an activity that is mediated by activation of nicotinamide adenine dinucleotide phosphate oxidase in adipocytes, as well as vascular smooth muscle and endothelial cells2,30,31. Consistent with previous in vitro findings, serum uric acid levels were reported to be positively associated with markers of oxidative stress levels in clinical settings13,14,15. However, such associations might actually reflect an association of XOR-derived ROS with oxidative stress level, since ROS and also uric acid are produced by an XOR-catalyzed reaction3,4, and plasma XOR activity has been shown to be positively associated with serum uric acid level10,11,12. In the present study, serum uric acid level, but not plasma XOR activity, showed a significant association with d-ROMs level (Table 3; Figs. 1, 2), suggesting that uric acid contributes to increased oxidative stress level independent of XOR activity through increased ROS production.

The association of serum uric acid level with d-ROMs level was prominent in females as compared to males in the present study (Fig. 3). Those results are consistent with previous reports showing that the association of serum uric acid level with oxidative stress level was more prominent in their female subjects13,14. Oxidative stress is considered to have a major role in the pathogenesis of lifestyle-related conditions, such as hypertension, nonalcoholic fatty liver disease, and metabolic syndrome, and also cardiovascular and cerebrovascular diseases32,33,34,35. On the other hand, previous meta-analysis findings showed that females with hyperuricemia had increased risk for development of the above-mentioned lifestyle-related diseases as compared to males with hyperuricemia36,37,38,39,40. Thus, we speculate that uric acid might be involved in the pathophysiology of lifestyle-related diseases by an association with increased oxidative stress, especially in females. Nevertheless, the underlying mechanisms involved in the gender-specific association of uric acid level in serum with oxidative stress level require further investigation.

The present study has several limitations. First, none of the enrolled subjects had hypouricemia, defined as a uric acid level in serum ≤ 2.0 mg/dL41, and few had hyperuricemia (n = 24), shown by a serum uric acid level > 7.0 mg/dL, as they were sequentially selected from individuals who voluntarily participated in a health examination. Although no significant association was noted between serum uric acid and BAP levels, there was a significant J-curve (nonlinear) association between serum uric acid and d-ROMs levels. However, we were not able to analyze those associations with stratification by hypouricemia or hyperuricemia. In addition, the association between serum uric acid and d-ROMs level seems to show a J-curve trendline at lower levels of serum uric acid, especially in males (Fig. 3). However, we were not able to analyze those associations by stratification with a low- or high-normal serum uric acid level, due to the low number of male subjects with a low-normal serum uric acid level. Second, the number of subjects with chronic kidney disease (CKD), defined as eGFR < 60 mL/min/1.73 m2, was few (n = 22) and none of the enrolled subjects had end-stage renal disease (ESRD), defined as eGFR < 15 mL/min/1.73 m2, or need for dialysis or renal transplantation. Although we previously found a positive association of plasma XOR activity with serum uric acid level in subjects who underwent health examinations as well as patients receiving hemodialysis treatments10,11,12, an inverse association of renal function with serum uric acid level independent of plasma XOR activity was also been revealed in our other results11,12, suggesting that the balance of serum uric acid level and plasma XOR activity might be different between individuals with and without CKD/ESRD. Third, we did not measure levels of antioxidants, such as vitamin C, glutathione, vitamin E, and polyphenol, thus were not able to comprehensively investigate the association between uric acid and antioxidant capacity including antioxidant levels. Fourth, uric acid administration testing was not performed, as the aim of the present study was to determine whether uric acid has an effect on oxidative stress level independent of XOR activity under physiological conditions. Therefore, differences in antioxidant and/or pro-oxidant effects exerted by intrinsic and exogenous uric acid were not clarified. Finally, because of the cross-sectional design, even though relationships were explored in predictive terms, the results cannot be interpreted to show causal relationships. A large-scale longitudinal study that includes subjects with hypouricemia, low-normal uricemia, hyperuricemia, or CKD/ESRD, as well as measurements of antioxidant levels and uric acid administration testing is needed to clarify the role of uric acid in regulation of oxidative stress.

In conclusion, the level of uric acid in serum showed a significant association with d-ROMs level in serum independent of plasma XOR activity in subjects registered in the MedCity21 health examination registry. That association was found to be more prominent in females, while there was no independent association with BAP level. These results suggest that uric acid contributes to increased oxidative stress by causing an imbalance between XOR-independent ROS generation and XOR-counterbalanced ROS scavenging, especially in females.

토론

이것은 인간의 요산 수치와 XOR 활성, 그리고 산화 스트레스 수준 사이의 관계를 조사한 최초의 연구입니다. 자발적으로 건강검진을 받은 피험자를 분석한 결과, 혈청 내 요산 수치는 혈장 XOR 활성과는 무관하게 BAP 수치와 유의미한 상관관계가 없는 것으로 나타났습니다(표 2). 반면에, 혈청 요산 수치는 혈장 XOR 활성도와 무관하게 d-ROM 수치와 유의미한 상관관계를 보였습니다(표 3; 그림 1, 2). 또한, 혈청 요산 수치와 d-ROM 수치 사이의 유의미한 상관관계는 남성보다 여성에서 더 두드러졌습니다(그림 3). 이 결과는 요산이 특히 여성에서 XOR 활성과는 독립적인 방식으로 생리적 조건 하에서 ROS 생성을 증가시킴으로써 산화 스트레스 수준을 증가시키는 데 기여한다는 것을 시사합니다. 한편, 생리적 조건 하에서 요산의 항산화 효과는 XOR 촉매 반응에 의해 생성된 ROS에 의해 상쇄될 수 있습니다.

산화 스트레스는 항산화 작용과 ROS 생산 사이의 관계가 교란됨으로써 발생하며, 산화 스트레스와 항산화 작용을 평가하기 위한 몇 가지 지표가 제안되었습니다. 항산화 작용과 산화 스트레스의 지표인 BAP와 d-ROMs에 대한 테스트는 실제로 자유 라디칼 연구에 사용할 수 있는 기술의 표준인 전자 스핀 공명(ESR)을 통해 얻은 결과로 실험적으로 검증되었습니다16,17. 또한, BAP 테스트는 철분 환원 능력과 혈장 항산화 테스트18,19와 같은 다른 항산화 능력 지표와 비슷한 결과를 제공하는 것으로 나타났으며, d-ROMs 테스트는 FOX 분석과 8-이소프로스타네20,21과 같은 다른 산화 스트레스 지표와 비슷한 결과를 제공하는 것으로 입증되었습니다. 따라서, 본 연구에서는 BAP와 d-ROMs 테스트를 통해 요산과 XOR 활동이 항산화 능력과 산화 스트레스 수준과 어떤 관련이 있는지 조사했습니다.

시험관 내 연구 결과에 따르면, 요산은 일중항산소, 퍼옥실 라디칼, 하이드록실 라디칼, 퍼옥시니트라이트와 같은 ROS를 제거함으로써 항산화 특성을 얻는 것으로 나타났습니다1,22. 또한, 건강검진을 받은 피험자14,15,23의 혈청 요산 수치가 항산화 잠재력의 지표와 양의 상관관계가 있는 것으로 보고되었습니다. 그러나, 이러한 상관관계는 요산 외에 ROS를 생성하는 XOR의 활성을 조정하지 않은 후 분석되지 않았습니다3,4. 혈장 XOR 활성을 공변량으로 포함한 다변량 회귀 분석의 현재 결과에서, 요산은 BAP 수치와 유의미한 상관관계가 없었습니다(표 2). 흥미롭게도, 요산이 인체에 미치는 항산화 효과는 외인성 투여와 같은 비생리학적 환경에서 수행된 시험에서 보고되었습니다. 예를 들어, 요산 또는 그 전구체인 이노신의 투여는 제1형 당뇨병 환자의 내피 기능을 개선하고24, 급성 허혈성 뇌졸중25 또는 다발성 경화증26 환자의 임상적 기능적 결과를 개선했습니다. 한편, 고요산혈증은 내피 기능 장애와 관련이 있는 것으로 밝혀졌으며27,28 급성 허혈성 뇌졸중 환자의 임상 기능적 예후를 예측하는 데 사용되었습니다29. 따라서, 본 연구 결과와 이전 연구 결과는 요산이 주로 외인성 투여와 같은 비생리적 조건에서 항산화 효과를 발휘하는 반면, 생리적 조건에서 XOR 촉매 반응에 의해 생성된 ROS에 의해 그 효과가 상쇄된다는 것을 시사합니다.

항산화 특성 외에도 요산은 지방세포, 혈관 평활근 및 내피세포에서 니코틴아미드 아데닌 디뉴클레오티드 포스페이트 옥시다제의 활성화에 의해 매개되는 활성산소(ROS)를 생성하는 등, 과산화물 음이온과 같은 활성산소를 생성하는 것으로 알려져 있습니다2,30,31. 이전의 체외 연구 결과와 일치하게, 혈청 요산 수치가 임상 환경에서 산화 스트레스 수치의 지표와 긍정적인 상관관계가 있는 것으로 보고되었습니다13,14,15. 그러나 이러한 연관성은 실제로 XOR-유래 ROS와 산화 스트레스 수준의 연관성을 반영할 수 있습니다. ROS와 요산은 XOR 촉매 반응에 의해 생성되기 때문이며3,4, 혈장 XOR 활성이 혈청 요산 수치와 양의 상관 관계가 있는 것으로 나타났습니다10,11,12. 현재의 연구에서 혈장 XOR 활성이 아니라 혈청 요산 수치가 d-ROMs 수치와 유의미한 상관관계를 보였으며(표 3; 그림 1, 2), 이는 요산이 ROS 생산 증가를 통해 XOR 활성과는 별개로 산화 스트레스 수치를 증가시키는 데 기여한다는 것을 시사합니다.

현재의 연구에서 혈청 요산 수치와 d-ROMs 수치 간의 상관관계는 남성보다 여성에서 더 두드러졌습니다(그림 3). 이러한 결과는 혈청 요산 수치와 산화 스트레스 수치의 연관성이 여성 피험자에서 더 두드러졌다는 이전 연구 결과와 일치합니다13,14. 산화 스트레스는 고혈압, 비알코올성 지방간 질환, 대사증후군과 같은 생활습관병의 발병 기전에서 중요한 역할을 하는 것으로 여겨지며, 심혈관 및 뇌혈관 질환에도 영향을 미치는 것으로 알려져 있습니다32,33,34,35. 반면, 이전 메타 분석 결과에 따르면, 고요산혈증이 있는 여성은 고요산혈증이 있는 남성보다 위에서 언급한 생활습관병에 걸릴 위험이 더 높습니다36,37,38,39,40. 따라서, 우리는 요산이 특히 여성의 경우, 산화 스트레스 증가와 관련되어 생활습관병의 병태생리학에 관여할 수 있다고 추측합니다. 그럼에도 불구하고, 혈청 내 요산 수치와 산화 스트레스 수치의 성별별 연관성에 관여하는 근본적인 메커니즘에 대해서는 추가적인 연구가 필요합니다.

이 연구에는 몇 가지 제한 사항이 있습니다. 첫째, 혈청 요산 수치가 2.0mg/dL 이하로 정의되는 저요산혈증 환자(n=0)는 없었고, 혈청 요산 수치가 7.0mg/dL 이상으로 나타나는 고요산혈증 환자(n=24)도 거의 없었습니다. 이들은 건강 검진에 자발적으로 참여한 사람들 중에서 순차적으로 선정되었기 때문입니다. 혈청 요산과 BAP 수치 사이에는 유의미한 연관성이 발견되지 않았지만, 혈청 요산과 d-ROMs 수치 사이에는 유의미한 J자 곡선(비선형) 연관성이 발견되었습니다. 그러나 저요산혈증 또는 고요산혈증에 따른 계층화를 통해 이러한 연관성을 분석할 수는 없었습니다. 또한, 혈청 요산과 d-ROMs 수치 사이의 연관성은 혈청 요산 수치가 낮을 때, 특히 남성에서 J자 곡선 경향을 보이는 것으로 보입니다(그림 3). 그러나, 혈청 요산 수치가 낮거나 정상인 남성 피험자의 수가 적기 때문에, 혈청 요산 수치가 낮거나 정상인 피험자를 대상으로 계층화 분석을 할 수 없었습니다. 둘째, eGFR이 60mL/min/1.73m2 미만인 만성 신장 질환(CKD) 환자 수는 적었고(n=22), 등록된 환자 중 eGFR이 15mL/min/1.73m2 미만인 말기 신장 질환(ESRD) 환자나 투석 또는 신장 이식이 필요한 환자는 없었습니다. 건강검진을 받은 사람과 혈액 투석 치료를 받는 환자에서 혈장 XOR 활성과 혈청 요산 수치의 양의 상관관계가 발견되었지만10,11,12, 혈장 XOR 활성과 무관하게 신장 기능과 혈청 요산 수치의 역상관관계가 다른 결과에서도 밝혀졌습니다11,12, 이는 혈청 요산 수치와 혈장 XOR 활성의 균형이 CKD/ES 환자군과 비환자군 간에 차이가 있을 수 있음을 시사합니다. RD. 셋째, 비타민 C, 글루타티온, 비타민 E, 폴리페놀과 같은 항산화 물질의 수준을 측정하지 않았기 때문에 요산과 항산화 능력(항산화 물질 수준 포함) 사이의 연관성을 종합적으로 조사할 수 없었습니다. 넷째, 본 연구의 목적은 생리학적 조건 하에서 요산이 XOR 활성과 무관하게 산화 스트레스 수준에 영향을 미치는지 여부를 확인하는 것이었기 때문에 요산 투여 테스트는 수행되지 않았습니다. 따라서 내인성 및 외인성 요산에 의한 항산화 및/또는 산화 촉진 효과의 차이는 명확히 밝혀지지 않았습니다. 마지막으로, 단면 설계를 사용했기 때문에 관계가 예측 가능한 방식으로 탐색되었더라도 그 결과를 인과 관계를 보여주는 것으로 해석할 수 없습니다. 요산혈증, 저요산혈증, 고요산혈증, 또는 만성신부전/말기신부전 환자들을 포함한 대규모 종단 연구와 항산화제 수치 측정 및 요산 투여 테스트가 필요합니다.

결론적으로, MedCity21 건강 검진 등록부에 등록된 피험자의 혈장 XOR 활성과 관계없이 혈청 내 요산 수치가 혈청 내 d-ROMs 수치와 유의미한 상관관계를 보였습니다. 이러한 상관관계는 여성에게서 더 두드러지는 것으로 나타났으며, BAP 수치와는 독립적인 상관관계가 없었습니다. 이 결과는 특히 여성에게서 요산이 XOR 독립적 ROS 생성과 XOR 균형적 ROS 소거 사이의 불균형을 야기함으로써 산화 스트레스 증가에 기여한다는 것을 시사합니다.

Subjects and methods

Study design

The MedCity21 health examination registry was initiated in April 2015 in a comprehensive manner to elucidate causes of various diseases occurring in adults, including cancer, diabetes mellitus, cardiovascular disease, cerebrovascular disease, mental disorders, dyslipidemia, hypertension, hyperuricemia, obesity, chronic respiratory disease, liver disease, digestive disease, gynecological diseases, and skin disease, for development of advanced diagnostic techniques, as well as treatment and prevention methods for affected individuals11,42,43. Those who voluntarily underwent heath examinations at MedCity21, an advanced medical center for preventive medicine established at Osaka City University Hospital (Osaka, Japan), were registered. The MedCity21 health examination registry protocol has been approved by the Ethics Committee of Osaka City University Graduate School of Medicine (Approval No. 2927). Written informed consent was obtained from all subjects and the study was conducted in full accordance with the Declaration of Helsinki. The present study protocol was approved by the Ethics Committee of Osaka City University Graduate School of Medicine (Approval No. 3684) and performed with an opt-out option, as explained in instructions posted on the website of the hospital.

Participants

Referring to the MedCity21 health examination registry of individuals examined between June 2015 and May 2017, the final 200 sequential subjects who participated in advanced comprehensive medical examinations designed to check the status of lifestyle-related diseases, such as hypertension, diabetes, dyslipidemia, visceral obesity, hyperuricemia, atherosclerosis, and cerebrovascular disease, were selected. For the present analysis, those being treated with an XOR inhibitor (n = 4), or uricosuric (n = 1) or insulin (n = 1) agents, or with missing data (n = 2) were excluded. As a result, 192 participants (91 males, 101 females) were enrolled as subjects in the present cross-sectional study.

Clinical assessments

Information for each subject regarding height, body weight, SBP, smoking and alcohol consumption habits, present and past illness, and use of medication was obtained. Body mass index was calculated as weight in kilograms divided by the square of height in meters (kg/m2). VFA values were obtained using a computed tomography device (Supria Grande, Hitachi, Ltd., Tokyo, Japan), as previously described44,45. Blood was drawn after an overnight fast, then biochemical parameters including serum uric acid level were analyzed using a standard laboratory method as part of the MedCity21 protocol and remaining blood samples were stored at − 80 °C. HbA1c level was determined as a National Glycohemoglobin Standardization Program equivalent value (%) using the conversion formula established by the Japan Diabetes Society46. eGFR was calculated using an equation designed for Japanese subjects, as previously described47. Serum immunoreactive insulin (IRI) level was measured with an electrochemiluminescence immunoassay (Roche Diagnostics K.K., Tokyo, Japan). The HOMA-IR index was calculated according to the following formula: fasting IRI (lU/mL) × fasting plasma glucose (mg/dL)/40548,49. The serum concentration of hs-CRP was measured using latex agglutination nephelometry (Siemens Healthcare Diagnostics, Inc., Marburg, Germany), as previously described50.

Serum BAP and d-ROMs levels

Freshly frozen serum samples were maintained at − 80 °C until the time of the assay. Levels of BAP and d-ROMs were assessed as markers of antioxidant potential and oxidative stress level, respectively, using a free radical elective eval‎uator system (FREE: Diacron International s.r.l., Grosseto, Italy) that included a spectrophotometric device reader, as previously described16,44,51. Briefly, for BAP, 50 μL of a ferric chloride solution was mixed with a thiocyanate derivative solution in a cuvette. Subsequently, 10 μL of serum was added and mixed, which resulted in a reduction of iron contained therein from a ferric to ferrous form by the actions of antioxidants. Color change in the cuvette was determined using a spectrophotometer at a wavelength of 505 nm and expressed as μmol/L. The intra- and inter-assay coefficients of variation in BAP level were 2.2% and 3.1%, respectively. As for d-ROMs testing, 20 μL of serum was mixed with an acid buffer solution (pH 4.8) in a cuvette. Subsequently, a chromogen for d-ROMS (reagent containing, N,N-diethyl-p-phenylenediamine, 20-μL) was added and mixed, resulting in oxidation of the chromogen substrate by free radicals. Color change in the cuvette was determined using a spectrophotometer at a wavelength of 505 nm and expressed as Carr units. The intra- and inter-assay coefficients of variation in d-ROMs level were 2.1% and 1.8%, respectively.

Plasma XOR activity

Freshly frozen plasma samples maintained at − 80 °C were used to determine XOR activity with a method recently established for assays of stable isotope-labeled [13C2,15N2] xanthine with LC/TQMS at Mie Research Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd, as we have previously described8,9. The intra- and inter-assay coefficients of variation of plasma XOR activity were 6.5% and 9.1%, respectively8.

Statistical analysis

Data are expressed as number or median. Values for HOMA-IR, hs-CRP, and plasma XOR activity were logarithmically transformed before performing multivariable regression analyses, due to the skewed distribution. Multivariable regression analyses were performed to determine whether the obtained uric acid level was independently associated with BAP or d-ROMs level after adjustment with various clinical parameters, including plasma XOR activity, as well as age, gender, and smoking and alcohol habits, and VFA, SBP, total cholesterol, HbA1c, HOMA-IR, eGFR, and hs-CRP levels. The non-linearity of the effect of uric acid level on BAP or d-ROMs level was included in the regression model. A two-factor interaction term (gender * uric acid) was also incorporated into the multivariable regression analysis model to assess the effect of gender difference on the relationship of uric acid level with BAP as well as d-ROMs level. The R software package, version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria), and Statistical Package for the Social Sciences, version 22.0 (PASW Statistics), were used for data analysis. All reported p values are two-tailed, and a p value of < 0.20 was considered significant for non-linear and interaction effects, as noted in previous studies52,53, while a p value of < 0.05 was considered to indicate significance for all of the other analysis results.

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