Analysis of the diagnostic efficiency of serum oxidative stress parameters in patients with breast cancer at various clinical stages

doi:10.1016/j.clinbiochem.2016.02.005

Clinical Biochemistry

Volume 49, Issue 9, June 2016, Pages 692-698

https://doi.org/10.1016/j.clinbiochem.2016.02.005 Get rights and content

Highlights

•
Both the hormone estradiol receptor and progesterone receptor positive rate were similar between two disease groups.
•
TOS and OSI were higher in patients than in healthy controls. TAS was lower.
•
From stages I to IV, serum TOS and OSI gradually increased, serum TAS decreased with a midpoint between stages II and III.
•
Boxplot analysis indicated that the OxS parameters were efficient for diagnosis.
•
The cut-off values for each OxS parameter were determined through ROC curve analysis.

Abstract

Background

Reactive oxygen species (ROS) are balanced through enzymatic mechanisms and exogenous antioxidants; imbalance results in oxidative stress (OxS). It is known that OxS plays an important role in the occurrence, development, and metastasis of breast cancer. The present study aimed to assess serum total oxidant status (TOS), total antioxidant status (TAS), and oxidant stress index (OSI) in patients at different clinical stages of breast cancer and to evaluate their diagnostic accuracy.

Methods

Serum TOS, TAS, and OSI were determined in 91 patients with breast cancer at different stages, 51 patients with benign breast tumors, and 35 healthy adults.

Results

Significant differences in serum TOS (F = 104.384, p = 0.000), TAS (F = 18.247, p = 0.000), and OSI (F = 62.598, p = 0.000) were observed among the 3 groups (benign breast tumor patients, breast cancer patients, and healthy women). Of the enrolled breast cancer patients, significant differences were also observed among different tumor stages, with TOS and OSI gradually increasing as the disease progressed, while TAS diminished. Receiver operating characteristic curve analysis revealed that the area under the ROC curve for OSI (AUC_OSI) was significantly higher than AUC_TAS (z = 2.344, p = 0.019) in distinguishing breast cancer from control groups (including disease control and the healthy control). The AUC_OSI (z = 4.700, p = 0.001) or AUC_TOS (z = 4.700, p = 0.001) was significantly higher than AUC_TAS in distinguishing breast cancer from the healthy control. The AUC_OSI (z = 5.907, p = 0.000) or AUC_TOS (z = 5.667, p = 0.000) was significantly higher than AUC_TAS in distinguishing benign breast tumors from the healthy control.

Conclusion

Oxidative stress parameters might serve as important indexes for monitoring breast cancer occurrence and progression. The combined evaluation of TOS, TAS, and OSI could be more beneficial for clinical assessment.

Introduction

According to the World Health Organization, breast cancer is one of the most common cancers in women, accounting for 16% of all female cancers, and its incidence is growing annually at a 2% rate [1]. Global cancer statistics for 2014 show that the morbidity and mortality of breast cancer account for 29% and 15% of those from all female tumors, respectively [2]. Breast cancer, therefore, is a serious concern for women's health and quality of life. However, approximately two-thirds of properly treated patients with early detected breast cancer can survive more than 20 years [3]. Thus, early detection is the basis of better prognosis and survival for breast cancer patients. A lot of tumor markers were associated with breast cancer, such as carcinoembryonic antigen, CA15-3, estradiol and progesterone receptors. But these markers have low performance for early diagnosis of breast cancer [4].

It is agreed on that intra-cellular oxidative damage is a general mechanism for cell and tissue injuries in vivo of cancer patients. Intra-cellular oxidative damage is mainly caused by oxidant, including free radicals and reactive oxygen species. The oxidant can react with unsaturated bonds of lipids in cell membrane and cause protein denaturation and damage in nucleic acid. In physiological conditions, antioxidants (enzymatic and non-enzymatic substances) can prevent and repair the damage of oxidant. Oxidative stress (OxS) appears through increasing oxidant generation and/or decreasing antioxidant levels in the target cells and tissues. In recent years, despite the recurrence and development of breast cancer induced by oxidative stress has gathered the attention of many researchers [5], [6], [7], [8], [9], most published reports have remained attentions on a single or several oxidant/antioxidant substances observed between plasma fluorescent oxidation products and breast cancer risk in proximate or distant samples [10]. Panis C, et al. found that OxS parameters were evaluated by plasmatic lipoperoxidation, carbonyl content, thiobarbituric reactive substances, nitric oxide levels, total radical antioxidant parameter, superoxide dismutase, catalase activities and GSH levels [11].

The lack of studies on overall OxS status is not encouraging for fully elucidating the relationship between breast cancer pathogenesis and overall serum OxS parameters [11], [12], [13]. The present study, therefore, aimed to explore the relationship between serum OxS status and breast cancer occurrence and development. We compared serum total oxidant status (TOS), total antioxidant status (TAS), and oxidant stress index (OSI) in patients with breast cancer at different stages, patients with benign breast tumors, and healthy women. We also compared the obtained serum data among enrolled breast cancer patients at different clinical stages. This information should help in assessing the diagnostic accuracy of OxS for breast cancer.

Section snippets

Subjects

This was a prospective diagnostic study. Ninety-one breast cancer patients (group A) and 51 benign breast tumor patients (group B), based on their presenting symptoms, from the Mianyang Central Hospital, Sichuan Province, China, as well as 35 healthy women (group C) from the same region. The patients were selected consecutively according to the inclusion and exclusion criteria from newly diagnosed patients treated at the hospital. The inclusion criteria included being female with no smoking

Background characteristics of the study population

The study population was selected between January 2010 and December 2013. All patients that fulfilled the eligibility criteria attended the study. Participant background demographics are shown in Table 1. The background data was similar between the groups. Chi-square (χ²) analysis showed that both the hormone estradiol receptor (χ² = 2.733, p = 0.098) and progesterone receptor (χ² = 1.641, p = 0.200) positive rates were not statistically different between the two disease groups (healthy control was not

Discussion

All aerobic organisms produce free radicals (FR) and reactive oxygen species (ROS) [20], [21]. FR and ROS commonly participate in a variety of normal physiological functions. However, when in excess, they can also attack biological macromolecules, leading to oxidative damage of cells and tissues [22], [23]. In order to counteract FR and ROS, living organisms utilize a peroxidation–antioxidant defensive system that relies on the formation of ROS/FR metabolites, and the rate at which these are

Conclusions

This study evaluated the diagnostic accuracy of serum oxidative stress parameters in breast cancer. The combined TOS, TAS, and OSI status of breast cancer patients can present the overall OxS status of a subject, which help us evaluate disease development and therapy's effects rather than provide a reliable method of direct diagnosis of breast cancer. Overall, oxidative stress parameters might serve as important indexes for monitoring breast cancer occurrence and progression. The combined

Disclosure statement

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by the People's Republic of China Ministry of Science and Technology (2006AA020905) (http://www.most.gov.cn/) and Biosino Bio-Technology and Science, Inc. (http://zhongsheng.shuoyi.com).

References (41)

O. Erel
A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation
Clin. Biochem.
(2004)
O. Erel
A new automated colorimetric method for measuring total oxidant status
Clin. Biochem.
(2005)
M. Harma et al.
Oxidative stress in women with preeclampsia
Am. J. Obstet. Gynecol.
(2005)
J.Q. Wu et al.
Free radicals, antioxidant defense systems, and schizophrenia
Prog. Neuro-Psychopharmacol. Biol. Psychiatry
(2013)
O. Marques et al.
Iron homeostasis in breast cancer
Cancer Lett.
(2014)
A.P. Wojtovich et al.
Optogenetic control of ROS production
Redox Biol.
(2014)
A. Jezierska-Drutel et al.
Role of oxidative stress and the microenvironment in breast cancer development and progression
Adv. Cancer Res.
(2013)
World Health Organization breast cancer: prevention and control....
R. Siegel et al.
Cancer statistics, 2014
CA Cancer J. Clin.
(2014)
C. Hammer et al.
Overview of breast cancer staging and surgical treatment options
Cleve. Clin. J. Med.
(2008)

C.M. Sturgeon

National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers

Clin. Chem.

(2008)

A.K. Rao et al.

Effects of Cu/Zn superoxide dismutase on estrogen responsiveness and oxidative stress in human breast cancer cells

Mol. Endocrinol.

(2008)

C.P. Rajneesh et al.

Lipid peroxidation and antioxidant status in patients with breast cancer

Singap. Med. J.

(2008)

S. Chariyalertsak et al.

Role of glutathione S-transferase omega gene polymorphisms in breast-cancer risk

Tumori

(2009)

N. Badid et al.

Oxidant/antioxidant status, lipids and hormonal profile in overweight women with breast cancer

Pathol. Oncol. Res.

(2010)

B. Melichar et al.

Effect of aromatase inhibitors on lipid metabolism, inflammatory response and antioxidant balance in patients with breast carcinoma

Anticancer Res.

(2009)

R.T. Fortner et al.

Plasma florescent oxidation products and breast cancer risk: repeated measures in the nurses' health study

Breast Cancer Res. Treat.

(2013)

C. Panis et al.

Differential oxidative status and immune characterization of the early and advanced stages of human breast cancer

Breast Cancer Res. Treat.

(2012)

J.F. Feng et al.

Serum total oxidant/antioxidant status and trace element levels in breast cancer patients

Int. J. Clin. Oncol.

(2012)

M. Mahajan et al.

Oxidative stress and its relationship with adenosine deaminase activity in various stages of breast cancer

Indian J. Clin. Biochem.

(2013)

Cited by (17)

Correlation of oxidative stress in patients with HBV-induced liver disease with HBV genotypes and drug resistance mutations
2018, Clinical Biochemistry
Citation Excerpt :
In addition, refreshing drinks were also stopped, including teas and coffees, as well as their extracts. Serum TAS level was determined by the method described by Erel et al. [12,13], with minor modifications. This assay relied on the ability of antioxidants in the sample to inhibit ABTS (2,2′-azino-di-3-ethylbenz-thiazoline sulfonate) from being oxidized into ABTS+ by the peroxidase metmyoglobin.
This study aims to explore the correlation of oxidative stress (OxS) in patients with chronic hepatitis B (CHB) and the disease severity with HBV genotypes and drug resistance mutations.
A total of 296 patients with CHB were enrolled into the study. PCR-reverse dot-blot hybridization was used to detect the HBV genotypes (B, C, and D) and the drug resistance-causing HBV mutant genes. In addition, the total oxidative stress (TOS) and total antioxidant status (TAS) were determined, and oxidative stress index (OSI) was calculated and compared.
Serum levels of TOS and OSI, the B/C ratio, and drug resistance mutation rate were increased along with the elevated disease severity degree (CHB<HBC<HCC) (P < 0.05), while the serum TAS level showed the opposite trend (though there was no statistical difference between patients at the CHB and HBC stages). Moreover, patients with drug resistance-causing HBV mutation had higher serum TOS and OSI levels, while lower serum TAS levels (P < 0.05), compared with patients without mutations. Furthermore, mutation site numbers were positively correlated with the disease severity degree (γ = 0.614, P < 0.001).
There is oxidative damage in patients with HBV-induced liver disease, and the damage degree is correlated with the HBV genotype and drug resistance mutation. Oxidative stress might be a useful indicator of the progression of HBV-induced liver disease in patients.
Serum and whole blood Zn, Cu and Mn profiles and their relation to redox status in lung cancer patients
2018, Journal of Trace Elements in Medicine and Biology
Citation Excerpt :
Gromadzińska et al. [29] also observed lower activity of SOD in lung cancer patients compared to the control group. Several studies have suggested that disruption in redox balance may deepen during cancer progression [11,26] or not [30,31]. In our study, levels of TAS, TOS and OSI, did not differ between patients with I–II and III–IV clinical stages, and levels of antioxidant enzymes did not differ either.
Disturbed redox status may be critical to lung cancerogenesis, however little research has been conducted on general changes in total redox status in lung cancer. Levels and activities of antioxidants, especially enzymatic ones, are related to trace element concentration. Trace element status is often disturbed in cancers, however no studies concerning the association between redox and trace element status have been performed for lung cancer. We hypothesized that disturbed redox status in lung cancer patients is partially determined by trace elements while their distribution amongst blood compartments may differ compared to healthy subjects.
Blood samples from lung cancer patients (n = 44) and control subjects (n = 44) were collected to assess redox and trace element status. Serum and whole blood Cu and Mn levels were determined with GF-AAS, and Zn–with F-AAS. In serum the total antioxidant status (TAS) was determined with the commercial kit TAS (Randox, UK), total oxidant status (TOS) was determined based on the method developed by Erel and the oxidative stress index (OSI) was calculated. Total protein (T-Prot), albumin (Alb), uric acid (UA) and total bilirubin (T-Bil) concentrations were measured with an auto-analyser (Konelab 20i, Thermoscientific, USA), SOD and CAT activity − with commercially available kits (Cayman, USA).
The level of TAS, T-Prot, Alb, T-Bil, the activity of SOD, the concentration of whole blood Mn as well as serum and whole blood Zn were lower while TOS, OSI, serum Cu levels and serum Cu:Zn ratios were higher in lung cancer patients compared to the control group. In the lung cancer group TAS correlated positively with Alb and UA, serum Zn and negatively with whole blood Mn. Additionally, SOD positively correlated with the whole blood Mn and Cu:Zn ratio, while CAT − negatively with the whole blood Cu:Zn ratio. In the lung cancer sub-group at clinical stage I–II, TOS additionally negatively correlated with whole blood Zn, and CAT negatively with serum Cu and Cu:Zn ratio. In advanced lung cancer, we found a positive correlation between TAS and serum Zn, and a negative one − with serum Cu:Zn ratio. We observed a similar correlation between endogenous non-enzymatic antioxidants and TAS in the control group, however considerably fewer correlations between trace elements and antioxidants were observed.
This study supports the hypothesis that disturbed redox status in lung cancer patients is linked with alterations in trace element status regarding Zn, Mn and Cu. Moreover, the type of biological fluid influences both − alterations in the metal profile and relationships with redox status parameters.
Investigation of dose-dependent effects of berberine against renal ischemia/reperfusion injury in experimental diabetic rats
2019, Nefrologia
Citation Excerpt :
Oxidative stress parameters including total antioxidant status (TAS) and total oxidant status (TOS) levels were determined by using an automatic biochemical analyzer (c800, Abbott, USA). Tissue TAS level was determined by Erel18,19. After this assay relied on the ability of antioxidants in the sample to inhibit ABTS (2,2′-azino-di-3-ethylbenz-thiazoline sulfonate) from being oxidized into ABTS+ by a peroxidase metmyoglobin.
Ischemia–reperfusion injury causes various severe morphological and functional changes in diabetic patients. To date, numerous antidiabetic and antioxidant agents have been used for treatment of the disease-related changes.
We aimed to examine effective therapeutic doses or doses of berberine against renal ischemia/reperfusion injury (IRI) in a streptozotocin (STZ)-induced diabetic rat model by histopathological and biochemical analysis.
Thirty male Sprague Dawley rats were treated with STZ injection for the development of diabetes, and divided into the following groups: STZ-induced diabetic group (STZ); IRI-induced diabetic group (STZ + IRI); 50 mg/kg berberine (BRB) treated diabetic group after inducing IRI (STZ + IRI + BRB₁); 100 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB₂); 150 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB₃). Bilateral renal ischemia model was applied for 45 min, then reperfusion was allowed for 14 days in STZ-induced diabetic rats. Renal injury was detected histopathologically. Blood urea nitrogen (BUN), creatinine and lactate dehydrogenase (LDH) levels were measured in serum using the ELISA method. Total antioxidant status (TAS) and total oxidant status (TOS) of renal tissue was studied by spectrophotometric assay. Oxidative stress index (OSI) was calculated as TOS-to-TAS ratio. Tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), Na⁺/K⁺-ATPase (sodium pump), and Ca²⁺-ATPase (calcium ATPase) enzyme levels were measured in tissues using the ELISA method. Anti-apoptotic Bax and pro-apoptotic Bcl-2 protein levels were detected by Western blot analysis. All data were evaluated statistically.
The highest histopathological score was detected in the STZ + IRI group compared to the other group. BRB administration at the doses of 100 mg/kg and 150 mg/kg markedly improved renal injury. BUN and creatinine levels significantly increased in the STZ + IRI group compared to the STZ group (p < 0.001). 100 mg/kg and 150 mg/kg BRB administration significantly decreased those levels (p < 0.01). The highest TOS and the lowest TAS levels were detected in the STZ + IRI group (p < 0.001). IRI markedly aggravated inflammation via increasing levels of TNF-α and CRP (<0.001), and caused apoptosis via inducing Bcl-2 protein, and suppressing Bax protein (p < 0.001). BRB administration at the doses of 100 mg/kg and 150 mg/kg showed anti-oxidant, anti-inflammatory and anti-apoptotic effects (p < 0.01). The LDH enzyme, was used as a necrosis marker, was higher in the STZ + IRI group than other groups. BRB administration at all of the doses, resulted in the decline of LDH enzyme level (p < 0.001). Ca²⁺-ATPase and Na⁺/K⁺-ATPase enzyme activities decreased in the STZ + IRI group compared to the STZ group (p < 0.001), while BRB administration at the doses of 100 mg/kg and 150 mg/kg significantly increased those of enzyme activities, respectively (p < 0.05).
Ischemia with diabetes caused severe histopathological and biochemical damage in renal tissue. The high doses of berberine markedly improved histopathological findings, regulated kidney function via decreasing BUN and creatinine levels, and rearranged intercellular ion concentration via increasing Na⁺/K⁺-ATPase and Ca²⁺⁻ ATPase levels. Berberine showed anti-oxidant, anti-apoptotic, and anti-inflammatory effects. According to these data, we suggest that berberine at the doses of 100 and 150 mg may be used as a potential therapeutic agent to prevent renal ischemic injury.
La reperfusión de la isquemia provoca graves cambios morfológicos y funcionales en los pacientes diabéticos. Hasta la fecha numerosos antidiabéticos y agentes antioxidantes han sido utilizados para el tratamiento de los cambios relacionados con la enfermedad.
El objetivo fue examinar las dosis terapéuticas efectivas o las dosis de berberina (BRB) frente a la insuficiencia renal por isquemia/reperfusión (IRI) en estreptozotocina (STZ) inducida por el modelo de rata por análisis histopatológico y bioquímico.
Treinta ratas machos Spraque-Dawley fueron tratadas con inyección de STZ para el desarrollo de diabetes, se dividieron en los siguientes grupos: grupo diabético inducido por STZ; grupo diabético inducido por IRI (STZ + IRI); grupo diabético tratado con 50 mg/kg de BRB después de la inducción de IRI (STZ + IRI + BRB₁); grupo diabético tratado con 100 mg/kg de BRB después de IRI (STZ + IRI + BRB₂), y grupo diabético tratado con 150 mg/kg de BRB después de IRI (STZ + IRI + BRB₃). Se aplicó un modelo de isquemia renal bilateral durante 45 min, luego se permitió la reperfusión durante 14 días en ratas diabéticas inducidas por STZ. La lesión renal fue detectada histopatológicamente. Los niveles de nitrógeno ureico en sangre (BUN), creatinina y lactato deshidrogenasa (LDH) se midieron en suero por el método ELISA. El estado antioxidante total (TAS) y el estado oxidante total (TOS) del tejido renal se estudiaron mediante un ensayo espectrofotométrico. El índice de estrés oxidativo (OSI) fue calculado como la relación TOS-a-TAS. El factor de necrosis tumoral alfa (TNF-α), proteína C reactiva (CRP), Na⁺/K⁺-ATPasa (bomba de sodio) y la Ca²⁺-ATPasa (calcio ATPasa) de la enzima se midieron los niveles en los tejidos mediante el método ELISA. Los niveles de proteína anti-apoptótica Bax y pro-apoptótica Bcl-2 se detectaron por el análisis de Western blot. Todos los datos fueron evaluados estadísticamente.
La mayor puntuación histopatológica fue detectada en el grupo STZ + IRI en comparación con el otro grupo. La administración de BRB en dosis de 100 y 150 mg/kg mejoró notablemente la lesión renal. Los niveles de BUN y creatinina aumentaron significativamente en el grupo STZ + IRI en comparación con el grupo STZ (p < 0,001). La administración de 100 y 150 mg/kg de BRB disminuyó significativamente esos niveles (p < 0,01). Los TOS más altos y los niveles más bajos de TAS se detectaron en el grupo STZ + IRI (p < 0,001). El IRI agravó notablemente la inflamación a través del aumento de los niveles de TNF-alfa y de PCR (< 0,001), y causó la apoptosis a través de la inducción de la proteína Bcl-2 y la supresión de la proteína Bax (p < 0,001). La administración de BRB con las dosis de 100 y 150 mg/kg mostró efectos antioxidante, antiinflamatorio y antiapoptóticos (p < 0,01). La enzima LDH que se usó como marcador de necrosis fue mayor en el grupo STZ + IRI que en los otros grupos. La administración de BRB en todas las dosis, dio lugar a una disminución del nivel de la enzima LDH (p < 0,001). Las actividades de las enzimas Ca²⁺-ATPasa y Na⁺/K⁺-ATPasa disminuyeron en el grupo STZ + IRI en comparación con el grupo STZ (p < 0,001, mientras que la administración de BRB en dosis de 100 y 150 mg/kg incrementó significativamente las actividades de las enzimas, respectivamente (p < 0,05).
La isquemia con diabetes causó graves daños histopatológicos y bioquímicos en el tejido renal. Las altas dosis de BRB mejoraron notablemente los hallazgos histopatológicos, la función renal regulada a través de la disminución de los niveles de BUN y creatinina, y la concentración de iones intercelulares reordenados mediante el aumento de los niveles de Na⁺/K⁺-ATPasa y la Ca²⁺-ATPasa. La BRB mostró efectos antioxidantes, antiapoptóticos y antiinflamatorios. De acuerdo con estos datos, sugerimos que la BRB en dosis de 100 y 150 mg se puede usar como un agente terapéutico potencial para prevenir la lesión isquémica renal.
Effects of Different Solvents on the Total Phenol Content, Total Flavonoid Content, Antioxidant, and Antifungal Activities of Micromeria graeca L. from Middle Atlas of Morocco
2024, Biochemistry Research International
Serum Bilirubin and Markers of Oxidative Stress and Inflammation in a Healthy Population and in Patients with Various Forms of Atherosclerosis
2022, Antioxidants
Redox and biometal status in Wistar rats after subacute exposure to fluoride and selenium counter-effects
2022, Arhiv za Higijenu Rada i Toksikologiju

View all citing articles on Scopus

View full text

Analysis of the diagnostic efficiency of serum oxidative stress parameters in patients with breast cancer at various clinical stages

Highlights

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Subjects

Background characteristics of the study population

Discussion

Conclusions

Disclosure statement

Acknowledgments

Clin. Biochem.

Clin. Biochem.

Am. J. Obstet. Gynecol.

Prog. Neuro-Psychopharmacol. Biol. Psychiatry

Cancer Lett.

Redox Biol.

Adv. Cancer Res.

Cancer statistics, 2014

CA Cancer J. Clin.

Overview of breast cancer staging and surgical treatment options

Cleve. Clin. J. Med.

National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers

Clin. Chem.

Effects of Cu/Zn superoxide dismutase on estrogen responsiveness and oxidative stress in human breast cancer cells

Mol. Endocrinol.

Lipid peroxidation and antioxidant status in patients with breast cancer

Singap. Med. J.

Role of glutathione S-transferase omega gene polymorphisms in breast-cancer risk

Tumori

Oxidant/antioxidant status, lipids and hormonal profile in overweight women with breast cancer

Pathol. Oncol. Res.

Effect of aromatase inhibitors on lipid metabolism, inflammatory response and antioxidant balance in patients with breast carcinoma

Anticancer Res.

Plasma florescent oxidation products and breast cancer risk: repeated measures in the nurses' health study

Breast Cancer Res. Treat.

Differential oxidative status and immune characterization of the early and advanced stages of human breast cancer

Breast Cancer Res. Treat.

Serum total oxidant/antioxidant status and trace element levels in breast cancer patients

Int. J. Clin. Oncol.

Oxidative stress and its relationship with adenosine deaminase activity in various stages of breast cancer

Indian J. Clin. Biochem.