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Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells

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• Published: 06 July 2016

Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells

• Lovro Ziberna, 
• Mitja Martelanc, 
• Mladen Franko & 
• Sabina Passamonti 

Scientific Reports volume 6, Article number: 29240 (2016) Cite this article

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Abstract

Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm‎ the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9–1.5 pmol mg−1 protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3–5 pmol mg−1 protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5–13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates and may thus be a dynamic factor of the endothelial function.

초록

빌리루빈은 

간 기능의 표준 혈청 생물학적 지표입니다. 

의외로, 

이는 심혈관 질환 위험과 역상관 관계를 보입니다. 

내피 기능 장애가 

심혈관 질환의 발생에 중요한 역할을 한다는 점을 고려할 때, 

혈관 내피에서의 빌리루빈 직접 분석은 이러한 관계에 대한 이해를 높일 수 있습니다. 

따라서 우리는 

내인성 세포 내 IXα-빌리루빈의 측정을 위해 

열 렌즈 분광 검출과 다이오드 어레이 검출을 결합한 

고성능 액체 크로마토그래피를 사용했습니다. 

IXα-빌리루빈의 동위체를 확인하기 위해, 우리는 전기분사 이온화 소스를 사용한 초고성능 액체 크로마토그래피와 고해상도 질량 분석기, 그리고 tandem 질량 분석 검출법을 사용했습니다. 쥐의 동맥 및 정맥 내피 세포(0.9–1.5 pmol mg−1 단백질)에서 빌리루빈을 측정했습니다. 인간 내피 세포주 Ea.hy926에서 세포 내 빌리루빈(3–5 pmol mg^(−1) 단백질)이 세포 외 빌리루빈 흡수 또는 내인성 빌리루빈 합성과 관련된 세포 내 효소인 헤모글로빈 산화효소-1(heme oxygenase-1)의 발현 증가에 의해 조절될 수 있음을 보여주었습니다. 

또한, 

빌리루빈의 세포 내 항산화 활성을 측정했으며, 

EC50은 11.4 ± 0.2 nM으로, 보고된 자유 혈청 빌리루빈 농도 범위(8.5–13.1 nM) 내에 있었습니다. 

빌리버딘은 

빌리루빈과 유사한 항산화 특성을 나타냈습니다. 

이러한 관찰 결과로부터, 내피 세포 내 빌리루빈이 진동하며

 내피 기능의 동적 요인일 수 있음을 추론합니다.

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Introduction

Bilirubin is a tetrapyrrole pigment present in various chemical forms in the blood, namely, conjugated with glucuronic acid (direct bilirubin), unconjugated bound to serum albumin (indirect bilirubin) and unconjugated-unbound (free bilirubin)1. Bilirubin is formed in cells by two sequential reactions, catalysed by heme oxygenase (biliverdin-producing, EC 1.14.99.3) and biliverdin reductase (EC 1.3.1.24). Approximately 250–300 mg bilirubin/day is formed in a normal adult. Most of bilirubin arises in the spleen from the catabolism of haemoglobin released form red blood cells. However, the pathway of heme catabolism is active in any kind of cells, supporting the turnover of heme, the cofactor of cytochromes and many other enzymes.

Bilirubin has antioxidant and anti-inflammatory activity and is inversely correlated with disease risk of the cardiovascular system, such as ischemic heart disease, hypertension, diabetes type II, metabolic syndrome and obesity, among others2,3. The vascular endothelium that lines the blood vessels plays a fundamental role in cardiovascular disease onset and progression4. Recent studies on the cellular composition of human and mouse tissue showed that endothelial cells are the most abundant cells in the heart—about 60 percent – thus implying to have an important role in heart physiology and pathology5. However, it is not known if the vascular endothelium is the transducer of the ‘bilirubin factor’ put in evidence by the aforementioned epidemiological studies. Therefore, the aim of our study was to provide the first ever, direct experimental proof for the presence and bioactivity of bilirubin in the vascular endothelium.

소개

빌리루빈은

혈액에 다양한 화학적 형태로 존재하는 테트라피롤 색소로,

글루쿠론산과 결합된 형태(직접 빌리루빈),

혈청 알부민에 결합된 비결합형(간접 빌리루빈),

그리고 비결합형-비결합형(자유 빌리루빈)으로 구분됩니다.1

빌리루빈은

헤모글로빈 분해 과정에서 헤모글로빈에서 생성되며,

헤모글로빈 분해는 헤모글로빈 분해 효소(헤모글로빈 분해 효소, EC 1.14.99.3)에 의해 촉매되는

두 단계의 연속 반응으로 이루어집니다.

정상 성인에서는

하루에 약 250–300mg의 빌리루빈이 생성됩니다.

대부분의 빌리루빈은

적혈구에서 방출된 헤모글로빈의 분해 과정에서 비장에서 생성됩니다.

그러나

헤모글로빈 분해 경로는

모든 종류의 세포에서 활성화되어

사이토크롬과 많은 다른 효소의 보조인자인 헤모의 회전을 지원합니다.

빌리루빈은

항산화 및 항염증 활성을 가지고 있으며,

심혈관 질환 위험과 역상관 관계를 보입니다.

예를 들어

허혈성 심장 질환, 고혈압, 제2형 당뇨병, 대사 증후군, 비만 등이 포함됩니다2,3.

혈관을 둘러싸고 있는 혈관 내피는

심혈관 질환의 발병과 진행에 근본적인 역할을 합니다4.

인간과 쥐 조직의 세포 구성에 대한 최근 연구는

내피 세포가 심장에서 가장 많은 세포(약 60%)를 차지한다는 것을 보여주며,

이는 심장 생리학과 병리학에서 중요한 역할을 함을 시사합니다5.

그러나 앞서 언급된 역학 연구에서 밝혀진

'빌리루빈 요인'의 전달체로 혈관 내피가 작용하는지 여부는 알려져 있지 않습니다.

따라서

본 연구의 목적은

혈관 내피에서 빌리루빈의 존재와 생물학적 활성을 직접 실험적으로 입증하는

최초의 증거를 제공하는 것입니다.

Results and Discussion

Is bilirubin an endothelial protective agent?

To assess the importance of bilirubin in endothelial protection from oxidative stress, we used the standard cell line EA.hy926 as a model of the human vascular endothelium. Cells were challenged with an oxidative stress, detected as a time-dependent increase of fluorescence emission by an intracellular probe. We assayed the effect of low concentrations of bilirubin (in the nM range) for 30 min, finding a concentration-dependent decrease in fluorescence. Data were plotted to quantify the antioxidant effect (Fig. 1) and obtain the EC50. The value found (11.4 ± 0.2 nM) was in the range of reported values of free serum bilirubin (8.5–13.1 nM)1. In such a range, the observed system is the most adaptable to changes (highest slope). This finding means that small changes in the extracellular bilirubin concentrations can be translated into substantially improved intracellular defence against oxidative stress. Biliverdin showed similar antioxidant properties as bilirubin, likely due to its conversion to bilirubin by biliverdin reductase6.

결과 및 논의

빌리루빈은 내피 보호제인가?

빌리루빈이

산화 스트레스로부터 내피 보호에 미치는 중요성을 평가하기 위해,

인간 혈관 내피의 모델로 표준 세포주 EA.hy926을 사용했습니다.

세포는

세포 내 프로브를 통해 시간 의존적 형광 발광 증가로 검출되는 산화 스트레스에 노출되었습니다.

빌리루빈의 저농도(nM 범위)를 30분 동안 처리한 후 형광 감소가 농도 의존적으로 감소함을 확인했습니다.

데이터를 그래프로 표시하여 항산화 효과를 정량화(그림 1)하고 EC50을 구했습니다.

측정된 값(11.4 ± 0.2 nM)은

혈청 내 자유 빌리루빈의 보고된 범위(8.5–13.1 nM)1 내에 있었습니다.

이 범위 내에서 관찰된 시스템은 변화에 가장 적응력이 높습니다(가장 높은 기울기).

이 결과는 세포외 빌리루빈 농도의 작은 변화가

산화 스트레스에 대한 세포 내 방어 능력의 현저한 개선으로 이어질 수 있음을 의미합니다.

빌리버딘은

빌리루빈 환원효소6에 의해 빌리루빈으로 전환되기 때문에

빌리루빈과 유사한 항산화 특성을 나타냈습니다.

Figure 1

Endothelial cellular antioxidant activity (CAA) of bilirubin and its synthetic precursor biliverdin.

Endothelial cells (Ea.hy926) were exposed to different concentrations of bilirubin or biliverdin (0.5, 2, 5, 10, 25, 50, 75 and 100 nM) in the albumin-free cell medium for a period of 30 min before the start of CAA assay. Results are expressed as means ± SEM from hexaplicates of 3 independent experiments.

빌리루빈 및 그 합성 전구체인 빌리버딘의 내피 세포 항산화 활성 (CAA).

내피 세포(Ea.hy926)는 CAA 분석 시작 전 30분 동안 알부민이 없는 세포 배지 내에서 빌리루빈 또는 빌리버딘(0.5, 2, 5, 10, 25, 50, 75 및 100 nM)의 다양한 농도에 노출되었습니다. 결과는 3개의 독립적인 실험에서 6회 반복 측정한 평균 ± SEM으로 표시되었습니다.

Is endothelial bilirubin measurable?

Based on these findings, we attempted at analysing intra-cellular free bilirubin, by an analytical HPLC method that replaced the standard diode array detector (DAD) detector with the ultra-highly sensitive thermal lens spectrometric detection (TLS). The quantification of intracellular bilirubin has never been attempted before, due to sensitivity limits of the current analytical methods. Only recently has a breakthrough in free bilirubin direct assessment in human and animal serum been accomplished, using a hyphenated HPLC-TLS technique1,7. The application of TLS detection instead of DAD has lowered the limits of both detection (LOD) and quantification (LOQ) by as much as 20-fold1.

Figure 2 shows the analysis of endogenous bilirubin in endothelial cells, based on MS and MS-MS fragmentation patterns, high-resolution mass (presented in Supplementary information) and retention time using HPLC-TLS, HPLC-DAD or UPLC-ESI-MS-MS system. Firstly, we used HPLC-TLS, due to its ultra-high sensitivity. Figure 2a shows the HPLC-TLS chromatogram of bilirubin standard solution containing three bilirubin isomers (III, IX, XIII-red line) and the endothelial cellular fraction (blue line). Co-elution of both HPLC peaks proved the presence of IXα bilirubin in the sample, devoid of unnaturally occurring III and XIII bilirubin isomers. Then, we applied an HPLC-DAD method, which enabled us to successfully perform the peak purity test by determining spectral homogeneity across the chromatographic peak, despite the fact that bilirubin concentration levels were at the LOQ (see Figure S1 in Supplementary information). The absorbance spectra obtained in both the region of eluted peak of standard bilirubin and in the peak present in the sample showed identical absorption pattern (Fig. 2b). Figure 2c shows UPLC-ESI-MS chromatogram of the endothelial cellular fraction, with peaks representing ions with m/z value of 583.3 [M−H+]−. This value was the same as that seen with bilirubin standard (Fig. 2d). Figure 2e shows that UPLC-ESI-MS-MS additionally confirm‎s the presence of intracellular bilirubin through comparison between the ion fragments pattern in the MS-MS spectra of bilirubin standard and the endothelial cellular fraction, both obtained after collision-induced fragmentation of pseudo-molecular ion (583.3 [M−H+]−); they showed an identical fragmentation pattern containing a typical bilirubin ion fragment corresponding to the m/z value of 285.1.

내피 세포 내 빌리루빈은 측정 가능할까요?

이러한 결과에 기반해, 우리는 표준 다이오드 어레이 검출기(DAD)를 초고감도 열 렌즈 분광 검출기(TLS)로 대체한 분석용 HPLC 방법을 사용하여 세포 내 자유 빌리루빈을 분석해 보았습니다. 세포 내 빌리루빈의 정량은 현재 분석 방법의 감도 한계로 인해 이전에 시도된 적이 없습니다. 최근에야 인간 및 동물 혈청에서 자유 빌리루빈의 직접 평가에 대한 돌파구가 HPLC-TLS 기술1,7을 사용하여 이루어졌습니다. TLS 검출을 DAD 대신 적용함으로써 검출 한계(LOD)와 정량 한계(LOQ)가 최대 20배까지 감소되었습니다1.

그림 2는 HPLC-TLS, HPLC-DAD 또는 UPLC-ESI-MS-MS 시스템을 사용하여 내인성 빌리루빈의 분석 결과를 보여줍니다. MS 및 MS-MS 분해 패턴, 고해상도 질량(보충 자료에 제시됨) 및 HPLC-TLS, HPLC-DAD 또는 UPLC-ESI-MS-MS 시스템의 유지 시간을 기반으로 합니다. 먼저 초고감도 특성 때문에 HPLC-TLS를 사용했습니다. 그림 2a는 세 가지 빌리루빈 이성체(III, IX, XIII-붉은 선)를 포함한 빌리루빈 표준 용액과 내피 세포 분획(파란 선)의 HPLC-TLS 크로마토그램을 보여줍니다. 두 HPLC 피크의 동시 분리는 시료에 IXα 빌리루빈이 존재함을 입증했으며, 자연적으로 발생하지 않는 III 및 XIII 빌리루빈 이성체는 검출되지 않았습니다. 그 다음, HPLC-DAD 방법을 적용하여, 빌리루빈 농도가 LOQ 수준(보충 자료의 그림 S1 참조)임에도 불구하고 크로마토그래픽 피크 전체에서 분광학적 동질성을 확인함으로써 피크 순도 검사를 성공적으로 수행했습니다. 표준 빌리루빈의 엘루트 피크 영역과 시료에 존재하는 피크에서 얻은 흡광도 스펙트럼은 동일한 흡수 패턴을 보여주었습니다(그림 2b). 그림 2c는 내피 세포 분획의 UPLC-ESI-MS 크로마토그램으로, m/z 값이 583.3 [M−H+]−인 이온을 나타내는 피크가 표시되어 있습니다. 이 값은 빌리루빈 표준과 동일했습니다(그림 2d). 그림 2e는 UPLC-ESI-MS-MS를 통해 빌리루빈 표준과 내피 세포 분획의 MS-MS 스펙트럼에서 이온 조각 패턴을 비교함으로써 세포 내 빌리루빈의 존재를 추가로 확인했습니다. 두 스펙트럼은 모두 가짜 분자 이온 (583.3 [M−H+]−)의 충돌 유도 분해 후 얻어졌으며, m/z 값 285.1에 해당하는 전형적인 빌리루빈 이온 조각을 포함하는 동일한 분해 패턴을 보여주었습니다.

Figure 2

Bilirubin determination in endothelial cells.

Endogenous bilirubin in endothelial cells (Ea.hy926) was identified by HPLC-TLS, HPLC-DAD or UPLC-ESI-MS-MS system. (a) HPLC-TLS chromatogram of bilirubin standard solution containing the three bilirubin isomers (III, IX, XIII - red line) and the endothelial cellular fraction (blue line). (b) HPLC-DAD chromatograms of bilirubin standard (10 nM, red line) and endothelial cellular fraction (blue line). (c) UPLC-ESI-MS chromatogram of analysed endothelial cellular fraction with peaks representing ions with m/z value of 583.3 [M−H+]−. (d) MS spectrum containing ion with m/z value of 583.3 [M−H+]− obtained after UPLC-ESI-MS analyses at retention time of bilirubin standard elution. (e) MS-MS spectrum containing ion with m/z value of 285.1 after UPLC-ESI-MS-MS of bilirubin standard and the sample of endothelial cellular fraction.

내피 세포에서의 빌리루빈 측정.

내인성 빌리루빈은 내피 세포 (Ea.hy926)에서 HPLC-TLS, HPLC-DAD 또는 UPLC-ESI-MS-MS 시스템을 사용하여 검출되었습니다. (a) 빌리루빈 표준 용액 (III, IX, XIII 이성체 포함 - 빨간 선)과 내피 세포 분획 (파란 선)의 HPLC-TLS 크로마토그램. (b) 빌리루빈 표준(10 nM, 빨간 선)과 내피 세포 분획(파란 선)의 HPLC-DAD 크로마토그램. (c) 분석된 내피 세포 분획의 UPLC-ESI-MS 크로마토그램으로, m/z 값 583.3 [M−H+]−를 갖는 이온의 피크가 표시됨. (d) 빌리루빈 표준 용출 시 UPLC-ESI-MS 분석 후 얻어진 m/z 값 583.3 [M−H+]− 이온을 포함하는 MS 스펙트럼. (e) 빌리루빈 표준 및 내피 세포 분획 샘플의 UPLC-ESI-MS-MS 후 m/z 값 285.1 이온을 포함하는 MS-MS 스펙트럼.

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The detector coupled to the HPLC system, i.e. either one based on TLS or a DAD, determines the performance of the methods. The HPLC-DAD method is limited by its sensitivity, which results in higher LOQ (3.5 pmol mg−1 protein) compared to HPLC-TLS (LOQ = 0.12 pmol mg−1 protein) and consequently in higher measurement error and therefore in lower accuracy of the results. For example, the LOQ of HPLC-DAD is about the same as changes in intracellular bilirubin levels observed in this study (3–5 pmol mg−1 protein).

In addition to endothelial cell culture experiments (EA.hy926 cells), we confirmed the presence of endogenous bilirubin in the endothelium isolated from both rat aorta and vena cava inferior. The values obtained were 1.53 ± 0.57 and 0.93 ± 0.14 pmol bilirubin mg−1 proteins (means ± SEM, n = 3), respectively.

HPLC 시스템에 연결된 검출기(TLS 기반 또는 DAD)는 방법의 성능을 결정합니다. HPLC-DAD 방법은 감도 한계로 인해 HPLC-TLS(LOQ = 0.12 pmol mg^(−1) 단백질)에 비해 LOQ(3.5 pmol mg^(−1) 단백질)가 높으며, 이는 측정 오차가 증가하고 결과의 정확도가 낮아집니다. 예를 들어, HPLC-DAD의 LOQ는 본 연구에서 관찰된 세포 내 빌리루빈 농도 변화 범위(3–5 pmol mg^(−1) 단백질)와 유사합니다.

내피 세포 배양 실험(EA.hy926 세포) 외에도, 쥐의 대동맥과 하대정맥에서 분리된 내피에서 내인성 빌리루빈의 존재를 확인했습니다. 측정된 값은 각각 1.53 ± 0.57 및 0.93 ± 0.14 pmol 빌리루빈 mg^(−1) 단백질(평균 ± SEM, n = 3)이었습니다.

Is endothelial bilirubin dynamically regulated?

The remarkable gain in sensitivity offered by combining HPLC with TLS detection enabled to address the question whether the vascular endothelium is a bilirubin-accessible compartment. Many clinical studies have shown the potential to modulate serum bilirubin values by either lifestyle modifications or pharmacological interventions targeting bilirubin transporters and enzymes, e.g. biliverdin reductase, heme oxygenase, UDP-glucuronosyltransferase (UGT1A1), organic anion-transporting polypeptide (OATP) and others. In turn, these changes can have an impact on individual cardiovascular disease risk8, as well as can be used as an early biomarker for the development of metabolic syndrome9. However, it is so far unanswered if bilirubin contents in the vascular endothelium can be modulated, which could result in a different capacity to withstand oxidative stress and limit cellular structural damage.

Thus, we designed two experiments aimed to assess dynamic changes in cellular bilirubin levels. In the first one, EA.hy926 cells were treated with 10 μM CoPPIX for 24 hours to induce the enzyme heme oxygenase-1 and thus increase cellular bilirubin synthesis. In the second one, the cells were kept in albumin-free cell media supplemented with 50 nM bilirubin for 30 min. The HPLC-TLS method enabled to measure increased intracellular bilirubin in both cases. Specifically, it was 3.14 ± 0.17, 4.34 ± 0.26 and 5.24 ± 0.51 pmol mg−1 protein (means ± SEM from triplicates of 6 independent experiments) under control conditions, heme oxygenase induction (P > 0.05) and bilirubin supplementation (P > 0.001), respectively. Though other technologies are needed to study the exact localization of bilirubin in the cells, it can be guessed that bilirubin can be present in: i) the cytosol, as either free or bound to intracellular proteins (e.g. glutathione-S-transferase A, previously known as ligandin); ii) cell membranes or iii) intracellular membrane compartments, due to its lipophilic character10. Whatever the case may be, these data corroborate the concentration-dependent antioxidant activity of bilirubin (shown in Fig. 1), either as a lipid peroxidation inhibitor or as a radical scavenger11,12.

내피 세포의 빌리루빈은 동적으로 조절되는가?

HPLC와 TLS 검출을 결합함으로써 얻은 감도 향상은 혈관 내피가 빌리루빈에 접근 가능한 부위인지 여부를 조사하는 데 기여했습니다. 많은 임상 연구에서 생활 방식 변경이나 빌리루빈 운반체 및 효소(예: 빌리버딘 환원효소, 헤모글로빈 산화효소, UDP-글루쿠로노실전달효소(UGT1A1), 유기 음이온 운반 폴리펩티드(OATP) 등)를 표적으로 한 약물적 개입을 통해 혈청 빌리루빈 값을 조절할 수 있음을 보여주었습니다. 이러한 변화는 개인의 심혈관 질환 위험에 영향을 미칠 수 있으며8, 대사 증후군 발병의 조기 바이오마커로 활용될 수 있습니다9. 그러나 혈관 내피 세포 내 빌리루빈 함량을 조절할 수 있는지 여부는 아직 명확히 밝혀지지 않았으며, 이는 산화 스트레스에 대한 저항 능력과 세포 구조 손상 제한 능력에 차이를 초래할 수 있습니다.

따라서 우리는 세포 내 빌리루빈 수준의 동적 변화를 평가하기 위해 두 가지 실험을 설계했습니다. 첫 번째 실험에서 EA.hy926 세포는 헤모글로빈 분해 효소 헤모글로빈 산화효소-1(heme oxygenase-1)을 유도하여 세포 내 빌리루빈 합성을 증가시키기 위해 10μM CoPPIX로 24시간 처리되었습니다. 두 번째 실험에서는 세포를 알부민이 없는 세포 배지(50 nM 빌리루빈 보충)에서 30분 동안 배양했습니다. HPLC-TLS 방법을 통해 두 경우 모두에서 세포 내 빌리루빈 농도가 증가한 것을 측정할 수 있었습니다. 구체적으로, 대조 조건에서는 3.14 ± 0.17, 헤모글로빈 산화효소 유도 시 (P > 0.05), 빌리루빈 보충 시 (P > 0.001) 각각 4.34 ± 0.26 및 5.24 ± 0.51 pmol mg^(−1) 단백질 (6개의 독립된 실험에서 3회 반복 측정치의 평균 ± SEM)이었습니다. 세포 내 빌리루빈의 정확한 위치를 연구하기 위해서는 다른 기술이 필요하지만, 빌리루빈은 다음과 같은 위치에 존재할 수 있다고 추측됩니다: i) 세포질 내 자유 형태 또는 세포 내 단백질(예: 글루타티온-S-트랜스퍼레이즈 A, 이전에 리간딘으로 알려져 있음)에 결합된 형태; ii) 세포막; 또는 iii) 세포 내 막 구획, 이는 그 지용성 특성 때문입니다10. 어떤 경우든, 이 데이터는 빌리루빈의 농도 의존적 항산화 활성(Fig. 1에 표시됨)을 뒷받침합니다. 이는 지질 과산화 억제제 또는 라디칼 제거제로서의 역할을 통해 나타납니다11,12.

Conclusions

Our data explain why increased HO-1 expression‎ level can lead to improved disease outcomes13. Moreover, this study enables to conclude that the protection against cardiovascular disease risk afforded by familial hyperbilirubinemia (Gilbert’s syndrome)14 is indeed mediated by an increased availability of the intracellular biliverdin/bilirubin redox pair, which improves the performance of the vascular endothelium in buffering reactive oxygen and nitrogen species15.

Given its anatomical size (ca. 240 m2 in a human subject)16, the contribution of the vascular endothelium to the physiological redox homeostasis is most prominent.

결론

우리의 데이터는 HO-1 발현 수준 증가가 질병 예후 개선으로 이어질 수 있는 이유를 설명합니다13. 또한 이 연구는 가족성 고빌리루빈혈증(길버트 증후군)이 심혈관 질환 위험으로부터 보호하는 메커니즘이 세포 내 빌리버딘/빌리루빈 환원 쌍의 가용성 증가를 통해 혈관 내피의 활성 산소 및 질소 종 완충 기능을 개선하기 때문임을 결론지을 수 있습니다14.

그 해부학적 크기(인간에서 약 240m²)16로 인해, 혈관 내피가 생리적 산화환원 균형에 기여하는 역할은 가장 두드러집니다.

Materials and Methods

Chemicals

Methanol and anhydrous dimethyl sulfoxide (DMSO), both HPLC grade, were obtained from Merck (Darmstadt, Germany). Fetal bovine serum (FBS), L-glutamine and penicillin-streptomycin solution were obtained from EuroClone (Milano, Italy); and 2′,7′-dichlorofluorescin diacetate (DCFH-DA), 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP), cobalt-protoporphyrin IX (CoPPIX), bilirubin and biliverdin were purchased from Sigma-Aldrich (Steinheim, Germany).

Phosphate Saline buffer (PBS): 200 mg/L KCl (Merck, Darmstadt, Germany); 200 mg/L KH2PO4 (Carlo Erba, Milano, Italy); 8000 mg/L NaCl; 1150 mg/L Na2HPO4 (Carlo Erba, Milan, Italy). Hank’s buffered saline solution (HBSS): 185 mg/L CaCl2 × 2H2O; 60 mg/L KH2PO4; 350 mg/L NaHCO3; 8000 mg/L NaCl; 47.88 mg/L Na2HPO4 (Carlo Erba, Milano, Italy); 100 mg/L MgCl2 × 6H2O; 1000 mg/L glucose (Sigma-Aldrich, Steinheim, Germany); 100 mg/L MgSO4 × 7H2O; 400 mg/L KCl (Merck, Darmstadt, Germany); the pH was adjusted to 7.4 using 0.1% HCl (Merck, Darmstadt, Germany). Ultrapure Milli-Q water (Merck Millipore, Billerica, MA) was used for the preparation of the solutions.

Bilirubin solutions

Bilirubin stock solution (5 mM) was prepared in DMSO. Then, it was diluted to 100 μM in 10 mM NaOH aqueous solution. Further diluted solutions were prepared using PBS. All dilutions were done in a dark room to avoid bilirubin photodegradation.

Cell culture

Human endothelial cell line Ea.hy926 (American Type Culture Collection, Rockville, USA) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM L-glutamine and 1 mM penicillin-streptomycin solution. Cells were grown in an incubator at 37 °C in a humidified atmosphere (95% air and 5% of carbon dioxide).

Cellular antioxidant activity assay

Endothelial cells EA.hy926 were seeded on 96-well plates (104 cells/well), containing 100 μL of complete DMEM/well and grown for 24 hours. Then, the cells were washed twice with PBS and incubated for 30 minutes with bilirubin or biliverdin, dissolved in 100 μL MGD solution (incomplete albumin-free DMEM, 1 mM L-glutamine, 50 μM DCFH-DA) at the following concentrations: 0 (control), 0.5, 2, 5, 10, 25, 50, 75 and 100 nM. After incubation, the solution was removed and the cells were washed twice with PBS. Then, the cells were treated with the peroxyl radical-generating reagent ABAP, at 5 mM in 100 μL Hank’s buffered saline solution (HBSS). The blank wells were filled with HBSS solution without ABAP. The fluorescence was measured at 535 nm with excitation at 485 nm every 5 min for one hour at 37 °C on a microplate reader (Synergy™ H1, Bio-Tek Instruments, Winooski, VT, USA). To quantify the cellular antioxidant activity, the CAA units (%) were calculated as follows:

where ∫SA is the integrated area under the sample fluorescence readings (with the blank subtracted) versus the time curve and ∫CA is the integrated area under the control curve.

Bilirubin uptake study

Endothelial cells (Ea.hy926) were grown in flasks (75 cm2) in completed DMEM for 48 hours to reach confluence. Monolayers were washed with PBS twice. Then, 500 μL of 50 nM bilirubin solution (dissolved in PBS) were added for 30 minutes. All experiments were performed in the dark room.

Heme oxygenase-1 (HO-1) induction

Endothelial cells (Ea.hy926) were grown in flasks (75 cm2) in completed DMEM for 24 hours. Then, they were incubated in CoPPIX solution (10 μM) dissolved in fresh completed DMEM for 24 hours before harvesting. CoPPIX is widely applied as a HO-1 inducer in various experimental models, especially in vitro cellular studies, where 3 μM CoPPIX induced maximal level of HO-1 expression‎ at 18 hours of incubation6.

Cell harvesting and sample preparation

Cells were washed with PBS twice, scraped in 500 μL of MeOH: DMSO (1:1, v/v) and collected into an Eppendorf vial. This protocol was repeated twice. The pooled harvest was ultrasonified for 1 min in an ice bath, rewarmed to room temperature and centrifuged at 12.000 rpm. To determine total protein concentration in the sample, we used Bradford assay on 20 μL of supernatant. The rest of supernatant was diluted with deionized water (1:1, v/v) to make the final sample prior to HPLC-TLS, HPLC-DAD or HPLC-ESI-MS analysis. In order to improve signal-to-noise ratio due to bilirubin’s low ionization efficiency and its low concentration in the 10 mL-injection loop of the UPLC system, some samples were concentrated by liquid-liquid extraction using chloroform (0.5 mL) prior to mass spectrometric analysis. The bottom chloroform phase was decanted to a new Eppendorf vial and the solvent was removed by a stream of nitrogen gas. The residue was redissolved in DMSO:H2O (1:1, v/v) to obtain a final volume of 300 μL (concentrated bilirubin solution).

Bilirubin analysis in cell samples

All mass spectrometry data were obtained by Waters Acquity ultra-performance liquid chromatograph (UPLC, Waters Corp., Milford, MA, USA) using the flow rate of 0.7 mL/min, the injection volume of 10 μL and the BEH C18 column (1,7 μm, 50 × 2,1 mm i.d.). Other chromatographic conditions for HPLC-TLS method were as previously published1.

The LC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer (Q-ToF Premier, Waters, Milford, MA, USA); analyses were made in negative ion mode using electrospray chemical ionization (ESI). The capillary voltage was 3.0 kV, while the sampling cone voltage was 20 V. The source and desolvation temperatures were 130 °C and 400 °C, respectively. The flow rate of nitrogen desolvation gas was 500 L/h. The acquisition range was between m/z 50 and m/z 1000 with argon serving as a collision gas at a pressure of 4.5 × 10−3 mbar in the T-wave collision cell. The tandem mass spectrometric analysis (MS-MS) was performed using collision energies from 15 to 30 eV to generate the product ion spectra that provided the best structural information. The data were collected in centroid mode, with a scan accumulation time of 0.2 s and an interscan delay of 0.025 s. The data station utilized the MassLynx v4.1 operating software. Accurate mass measurements (Supplementary Information Figure S2) were obtained with an electrospray dual sprayer using the reference compound leucine enkephalin ([M + H+]+ = 556.2271) at a high mass resolution of approximately 10000 full width of the peak at half its maximum height (FWHM) UPLC-MS spectrum in the Fig. 2c was obtained after a sample (from the control group) 5-fold pre-concentration (by solvent evaporation under reduced pressure), thus in the 3–6 min time range some peaks appear due to bilirubin degradation during sample concentration. Sample concentration prior analysis was needed, since the bilirubin signal intensity (m/z of 583,2) in the analysed sample (from the control group) was almost insignificant (under LOD), as shown in Supplementary Information (Figure S3).

Limit of detection (LOD) and limit of quantification (LOQ) were determined using the peak representing blirubin based on a visual determination of a peak-to-peak signal-to-noise ratio of at least 3:1 and 10:1, respectively.

Animals

Adult male Wistar rats weighing 330–350 g (5 months of age) were housed under standard laboratory conditions in a temperature-controlled environment (22 ± 1 °C, 60% humidity) maintained on a 12-h light/dark cycle. All animal procedures and study protocols were approved and conducted in accordance with the permission issued by the Veterinary Administration of the Republic of Slovenia (permit SI-No. U34401-4/2014/4, given to Institute of Pharmacology, Faculty of Medicine, University of Ljubljana), which conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Blood vessel isolation

Three rats were killed by cervical dislocation and bled. From each animal both aorta and vena cava inferior were dissected from the thorax, cleaned of connective tissue and periadventitial fat. To remove any remaining blood, blood vessels were rinsed with cold PBS three times (by using a syringe) and were left for additional 5 minutes in the 50 mL beaker filled with the cold PBS solution under dark conditions. To expose the complete endothelium surface, all blood vessels were carefully opened by a longitudinal cut. The endothelial cells were gathered by gentle abrasion with a clean scalpel blade from the edges to the center of the blood vessel. Detached cells were immediately collected in 100 μL of MeOH: DMSO (1:1, v/v) into an Eppendorf vial. The pooled harvest was ultrasonified for 1 min in an ice bath, rewarmed to room temperature and centrifuged at 12.000 rpm. To determine total protein concentration in the sample, we used Bradford assay on 20 μL of supernatant. The rest of supernatant was diluted with deionized water (1:1, v/v) to make the final sample prior to HPLC-TLS.

Additional Information

How to cite this article: Ziberna, L. et al. Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells. Sci. Rep. 6, 29240; doi: 10.1038/srep29240 (2016).

References

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