abstract: # Background
  .
  The majority of South African aging population are women, who spend late adulthood experiencing natural menopause. Despite the government spending billions of rand on different services for ageing women, menopausal challenges to African women still receive little attention.
  .
  # Objectives
  .
  The aim of the study was to explore and describe the perceptions of African women regarding natural menopause, in order to propose recommendations for health and social support systems for women in Mamelodi, Tshwane district.
  .
  # Method
  .
  A qualitative, exploratory, descriptive and contextual design was used to conduct the study. The population of the study consisted of menopausal women, between the ages 45 and 60 years or more, visiting the clinics for collection of chronic medication and other health assessment. Individual face-to-face interviews were conducted, using a semi-structured interview guide to collect data. Tesch's method of qualitative data analysis was used in the study.
  .
  # Results
  .
  The main theme that emerged from the study was 'attitude toward menopause', which was supported by cultural beliefs and experience. The African menopausal women expressed the importance of health support systems that will meet their needs within their context.
  .
  # Conclusion
  .
  Women's health programs and educational health information at facilities should include menopausal education to promote and improve health of all African menopausal women during their adulthood. There is a need to establish a women's health support group network within communities to share menopausal experiences with peers. The training and education curriculum of healthcare providers should include detailed menopause in order to provide comprehensive, congruent care.
author: Gloria N. Makuwa; Steppies R. Rikhotso; Fhumulani M. Mulaudzi**Correspondence to:** Gloria Makuwa, **Email:**<email@example.com>**Postal address:** 1499 Kaallaagte Street, Pretoria 0186, South Africa[^1]
date: 2015-12-17
references:
title: The perceptions of African women regarding natural menopause in Mamelodi, Tshwane district

# Introduction

Menopause is a normal, natural phenomenon for all ageing women, irrespective of culture or country of origin (Ramakuela et al. 2014:1). Rabiee, Nasirie and Zafarqandie (2014:4) explained menopause as a complex and multidimensional biological, social and psychological phenomenon for women between, on average, the ages of 45 and 60 (or more) years.

To promote women's health during middle age and beyond, it is necessary to understand that menopause is a natural ageing process that all women undergo because of reduced production of reproductive hormones, oestrogen and progesterone (Lobo 2012; Rahman, Salehin & Iqbal 2011:188). There are a multitude of factors to be considered for menopause besides the women's age; these include biological, socioeconomic and lifestyle factors. Rabiee et al. (2014:1) argued about the factors which affect the transitional period in a woman's life and its relation to sexual desire in some women.

According to Norwitz and Schorge (2010:63), women experience menopause in a universal manner, as all bodily signs and symptoms are influenced by factors such as an ageing endocrine system leading to decline in ovarian function, a decrease in oestrogen that causes hot flushes, night sweats and vaginal dryness. However, the actual experience and attitude of women on menopause is individualistic. The attitude of women is ominously influenced by sociocultural factors – race, culture and ethnicity – as well as multicultural factors (Rapaport 2015:1). Agwu, Umeora and Ejikeme (2008:217) argued differently that not all women experienced menopause the same way, as their perceptions depend on their individual and sociocultural backgrounds.

In multicultural South Africa, health beliefs and practices vary within different communities. Menopause is viewed by some African women as the freedom from monthly menstruation and the transition period toward permanent infertility (Helman 2007:47; Ramakuela et al. 2014:2). Helman (2007:48) added that in the Zulu culture, for example, women who are in menopause are viewed as clean and can enter into the field or cows' kraal without ruining the crops or causing sickness to the cattle. Ande et al. (2011:300) and Ozuzu-Nwaiwu (2007:34) support the view that most African women's cultures accept menopause as a natural ageing process caused by a supernatural effects, resulting in African women rarely refusing hormone replacement therapy (HRT) (Ande et al. 2011:300; Ozuzu-Nwaiwu 2007:34), because of inadequate reproductive health-related knowledge about the phenomenon.

Some African women's cultures perceive menopause as being a negative life event and a 'disease-entrapment process' resulting from cessation of menstruation, causing illness or poison in the woman's body system. Limited literature has been conducted in the South African context to understand the perceptions of African women regarding menopause (Ozuzu-Nwaiwu 2007:35). As a result, the study was conducted to explore and describe the perceptions of African women regarding natural menopause in Mamelodi, Tshwane district.

## Problem statement

Women in multicultural South Africa perceive natural menopause differently as it is influenced by geographical location, cultural beliefs, socialisation, female reproductive health knowledge deficit and societal practices. Ramakuela et al. (2014:1) alluded that menopause in a rural village of Limpopo Province is determined by cultural beliefs because menopause is considered to be a taboo and/or sensitive discussion topic, meaning that women remain silent for fear of embarrassment (Agwu et al. 2008:220). Mustafa and Sabir (2012:176) revealed that other cultures, for example, Western women, report menopausal symptoms and receive assistance and support as they have reproductive health-related knowledge, including regarding ageing process. The cultural silence held by some African menopausal women may result in health-related problems because of inadequate knowledge. Approximately 51% of the South African population are women, with an average age of 55.2 years (Statistics South Africa 2012:3) and it is of great concern that few studies have been conducted that focus on understanding the cultural perceptions of South African women regarding reproductive health knowledge on natural menopause.

Currently, African women who are experiencing menopause reported health problems related to menopause only if they had a serious illness accompanied by unexplained pains, irritability, unusual emotional behaviour or unexplained signs and symptoms. The failure of some menopausal women to openly discuss and share information and reproductive health-related knowledge regarding natural menopause may lead to misunderstandings about menopause. There have been only a few studies done which are related to the perceptions of women regarding natural menopause.

### Aim and objective of the study

The aim of the study was to explore and describe the perceptions of women regarding natural menopause in order to propose recommendations for healthcare providers to provide reproductive health-related knowledge regarding human developmental stages for all women in Tshwane district.

## Definition of concepts

### Perceptions

The receiving, selecting, organising and interpreting of sensory information or stimuli by combining them with the results of previous experience to produce a meaningful picture of one's world (Lantos 2010:388). It refers to how natural menopausal African women describe the experiences and feelings of being in the transitional period of their life, between the ages 45 to 60 years.

### African woman

An 'African woman' is an adult woman who bears children and has a specified occupation or role in a family context such as a wife and/or mother or grandmother (Stevenson & Waite 2011:1659). In this study, it refers to an African woman between the ages of 46 and 55, experiencing natural menopause and residing in Mamelodi, Tshwane district. The African woman's lifestyle and way of life is to focus on child bearing and caring for their large family (Hall et al. 2007:107).

### Menopause

This refers to the condition designed by nature and regarded as a normal ageing process (Ramakuela et al. 2014:1), whereby the woman's monthly menstrual periods ceases. It is normally diagnosed retrospectively a year following secondary amenorrhoea in woman of 45 years of age or older.

### Significance of study

The findings of this study may assist in providing African women with health-related knowledge regarding menopause and contribute to the body of knowledge in nursing and other health professionals to provide comprehensive and congruent care to menopausal women of different African cultures. This could be achieved by sharing information through training institutions for the inclusion of the curriculum on women's health, focusing on menopause. The findings will further inform the authorities to provide assistance to ageing African women for the establishment of health support groups in the community of Mamelodi in Tshwane district, where menopausal women share their menopausal experiences with peers.

# Research design

The research design is the overall plan for conducting research that will be applied in an attempt to address the research questions, including specifications for enhancing the study's integrity (De Vos et al. 2011:309). A qualitative, explorative, descriptive and contextual design was used.

## Research approach and method

The design enhanced a systematic and interactive approach exploring life experiences of human participants to attach meaning (Polit & Beck 2012:16). The explorative aspect of the study was to explore the dimensions and relationships between the phenomena that were studied (Polit & Beck 2012:727); and the existing perceptions of African women regarding natural menopause were discovered through interviews and observations.

Descriptive study, according to Botma et al. (2010:194) is a study that is based on the general premises of naturalistic inquiry. The perceptions of African women regarding natural menopause were described within the context of the study setting. Contextual research is based on findings valid within the time, space and value context in which the study was being conducted (Polit & Beck 2012:732), in this case, in Mamelodi, Tshwane district.

Data were collected from African women who were at the menopausal stage of between the ages of 45 and 60 years, using semi-structured, individual interviews in Mamelodi, Tshwane district.

### Context of the study

This article is written based on a mini-dissertation submitted for a Master's degree at the University of Pretoria. The study was conducted in two Mamelodi clinics in Tshwane district.

### Population, sample and sampling technique

African menopausal women were recruited during day clinic visits for minor ailments, routine body check-ups and for chronic medication collection, through the assistance of the clinic professional nurses at the setting. The purposive-convenience sampling technique was used to sample the participants (Botma et al. 2010:125). It is purposive as the researcher selected a representative sample of African menopausal women, namely, those between the ages of 45 and 60 years old.

The participants were purposively handpicked from the population (patients at the clinic) based on the researcher's knowledge and judgement. It is also convenient, as the African menopausal women selected were only those who were at the clinic on the day of data collection, as assisted by the clinic professional nurses. The sample was drawn from the population according to the sample inclusion criteria, as described below.

### Inclusion criteria

The inclusion criteria refer to those criteria that specify the population characteristics to be included in the study, that is, the eligibility criteria (Polit & Beck 2012:274). The sampling criteria for this study were:

-   An ageing woman between 45 and 60 years old.

-   Must have had no menstrual periods for the past 12 months.

-   Must be attending the clinic services in Mamelodi, Tshwane district.

-   Must reside in Mamelodi, Tshwane district.

-   Must be willing to share their perceptions regarding menopause with the researcher voluntarily and without any coercion.

### Data collection

The semi-structured, one-to-one individual interview method was used in the study to produce rich data as the participants were able to reflect and describe their perceptions regarding natural menopause in their own words.

### Accesses to participants

The majority of the women were at the clinic to collect their chronic medication and, coincidentally, they met the inclusion criteria for the study. The main questions were asked by the researcher as guided by the interview guide and some probing questions followed. A digital recorder was used to record the interviews; and field notes were taken for non-verbal cues through observation by the researcher. Data were collected until data saturation was reached with the 18th participant, that is, when the menopausal women were providing information similar to the information already gathered from previous participants.

### Data treatment

Data analysis is a process of bringing order and meaning to a large amount of data into smaller, manageable segments that can be retrieved and revised (De Vos et al. 2011:397). Data were analysed according to Tesch' s (1990) eight steps of data analysis, as follows:

1.  All data collected through interviews, digital recorder and observations were transformed into written text with the date, time and the place where the interview took place. Field notes were also typed and labelled.

2.  The researcher read through the data several times to obtain a general sense of the information and to reflect on its overall meaning.

3.  Several transcripts were read through and highlighted or segments (phrases) in them.

4.  A list was compiled of all the topics that came to the researcher's mind.

5.  The researcher used the compiled list to analyse the transcripts by looking for segments (phrases) from the transcripts that fit the topics.

6.  All the segments that fitted a particular topic were put together and were given descriptive names as subthemes.

7.  The subthemes were sorted and grouped together, then given descriptive names as themes.

8.  Digital recorder interviews were transcribed verbatim, then analysed and clustered into themes, categories and subcategories.

# Ethical considerations

The study obtained ethical clearance from the University of Pretoria's Ethics Committee (protocol number 32/2014), the Ethical Committee of the Gauteng Department of Health, Tshwane district and the Nursing management of the two clinics in Mamelodi, Tshwane district. The recruited participants were provided with information leaflets and the researcher explained the aims, objectives and significance of the study. They were reassured that no harm would be experienced, either emotionally, physically or psychologically. The participants were given informed consent forms to sign voluntarily and without coercion. Confidentiality and anonymity were maintained throughout the report (De Vos et al. 2011:116; Polit & Beck 2012:177) and transcripts and recordings were kept in a locked safe by the researcher.

# Trustworthiness

The following criteria were used and applied throughout the study to ensure trustworthiness: credibility, dependability, transferability and confirmability (Botma et al. 2010:232):

-   **Credibility** was enhanced by using member checking and peer examination. The researcher conducted semi-structured, one-to-one individual interviews until data saturation was reached, using digital recorder and field notes to capture accurate data. The data collected were continuously checked with the participants to confirm that transcriptions were true descriptions and interpretations of every participant's own perception and experience. Field notes and the digital recording were given to the supervisor to check the allocated themes.

-   **Dependability** refers to the findings that will not change if a similar study with the same participants and in a similar context were done at a different time (Polit & Beck 2012:585). The researcher used individual semi-structured, one-to-one individual interview method for all the participants. A digital recorder and field notes were used to ensure an audit trail. Systematic documentation was created by the researcher for the purpose of the audit trail that was available and accessible to the supervisors as proof of all the decisions made throughout the study. The data were analysed and an agreement was reached by the researcher and supervisors with regard to identification of themes.

-   **Transferability** was ensured by the researcher by providing thick description of context, the type of sampling method and the characteristics of participants, data collection and analysis.

-   **Confirmability** refers to the objectivity or neutrality of the data; there must be an agreement between two or more independent people regarding the data's relevance and its meaning (Polit & Beck 2012:585). The researcher and the supervisor reduced data to themes after consensus had been reached.

# Results and discussion

The main theme that emerged from the study was: attitude toward natural menopause.

## Attitude toward natural menopause

This theme exposed the attitude of women regarding natural menopause based on their culture, experience and knowledge. Adequate knowledge and better understanding about menopause by women leads to a positive attitude toward this natural stage in life. If there is misunderstanding about reproductive health-related knowledge on menopause, some women may misunderstand the feeling of being in the menopausal state, such as mood swings and irritability, resulting in inadequate acceptance of this normal phenomenon in ageing women and may thus be perceived in a negative light (Adewuyi & Akinade 2010:1778).

### Positive attitude

Most participants in the study regarded menopause as a natural process of ageing and life changes. The participants understood natural menopause as a cessation of the menstrual circle, resulting in no more reproduction or falling pregnant. The study conducted by Im et al. (2008:548) revealed that most women had a positive attitude toward natural menopause.

African women above 60 years who experienced natural menopause for a longer period, perceived it positively. According to Ramakuela et al. (2014:2), menopause was accepted as a normal phenomenon and menopausal women felt happy about the post-reproductive life stage. Some of the participants said:

> 'I think menopause is the time when you become old and you are no longer going to fall pregnant again. It is a natural change of life and ageing. We are living a good life like children. You no longer have a problem of buying some pads. I really enjoy this stage. You can sleep without clothes and not being disturbed.' (File C/Participant 7, 60 years old)
>
> 'I feel alright with the stage of menopause. I must admit that now is the time I did not start of being in the stage of menopause \[*sic*\] but I started there, way back, as a lady. Now is the time to be in this stage because everything has its time and I cannot be ever young.' (File C/Participant 17, 58 years old)
>
> 'I am growing old. The age is catching up with me but I am just happy because I do not have my periods or buy any pads.' (File D/Participant 1, 63 years old)

As noted from the quotes above, African women with experience, knowledge and who are above 60 years of age accept natural menopause as a normal ageing process. Sociocultural beliefs had no influence on the freedom to discuss natural menopause openly with the researcher, who was not only a woman but also above 50 years of age and within the class of African menopausal women. The African women discussed natural menopause freely without any signs of embarrassment and accepted the physiological changes occurring in their bodies without complaints.

### Negative attitude

Although most of the participants accepted menopause as a natural process of ageing, some participants who were below the age of 60 were negative, due to about the changes occurring in their bodies after cessation of menstruation. These women highlighted their lack of knowledge about natural menopause because of cultural beliefs, which restricted them from talking about it, as it is regarded as taboo. One participant said:

> 'I do not have enough information about menopause. It is a difficult stage because you do not have the experience and knowledge of what to expect. I do not understand why there are changes in my body or life as whole.' (File D/Participant 11, 52 years old)

The quote above indicates that the participant (52 years old) did not understand the changes occurring in her body and lacked knowledge about the physiological processes associated with female ageing. Below, the quote from another participant:

> 'At least, when you were menstruating every month, it prevented you from being ill. But now, my menstruation is no longer coming out and causes a lot of diseases. I am person who is having a lot of blood. Since I stopped to have my monthly period, my blood pressure is high. It can be a problem; you can maybe have … womb cancer. There are lots of diseases which can occur in your body.' (File C/Participant 15, 49 years old)

Participants below 60 years of age believed that the 'blood that is not coming out' (File D/Participant 11, 53 years old) accumulates in their body and cause health problems. The women presented the following signs and symptoms as caused by menopause: headache, backache, painful joints and generalised body pain. Pain was the most common menopausal symptom linked with blood 'that does not come out', as shown in the quote below. One of the participants said:

> 'I think it is this blood that does not flow, that does not come out. I do not know where this blood goes. I am thinking that maybe it is accumulation of blood in the body that causes pains because the blood does not manufacture babies any longer and no more functioning \[*sic*\].' (File D/Participant 11, 53 years old)

Some of the participants in the study related the body changes to changes in their hormones and retained blood resulting in weight gain and skin pigmentation, as revealed below:

> 'My skin and body size have changes. The face is dark and wrinkled and not shiny like when I was young. My skin is dry and itching even if I have washed; I think is the hormones and ageing.' (File D/Participant 4, 55 years old)
>
> 'I gained weight. My face has pimples because of the "blood that does not come out."' (File C/Participant 11, 53 years old)

The explanation above indicates that some women lack knowledge about menopause and have contradictory sentiments. This was supported by the study conducted by Mustafa and Sabir (2012:175), who stated that women believe menopause is a natural condition that affects them in a negative manner. The study conducted by Agwu et al. (2008:219) alluded to the fact that some women believed that abstinence from intercourse precipitates menopause and heralds its symptoms.

A widowed participant said:

> 'I think menopause is to grow up but my elder sisters, who are older than me, are still menstruating. They are having their husbands. So, I think when you are menstruating, you sleep with a man. I am not sleeping with a man, that it is why menstruation has stopped. The blood stays in your body makes you sick, it must come out every month. It stays under the stomach and at the back and make you feel pains.' (File D/Participant 4, 46 years old)

This indicates that some of the women below 60 years of age do not understand the causes of menopause because of limited experience and inadequate reproductive health-related knowledge during their menopausal stage and education development regarding menopause and procreation. It is supported by the quote below, which implies that menopause was regarded as taboo to discuss with an elder; there was no sharing of knowledge and experience.

One participant said:

> 'I have so many complaints and my mother has six children, but I do not remember her complaining about heat and other diseases. What makes me feel bad is that I did not have my own children.' (File D/Participant 12, 48 years old)

The study revealed that the attitude of some African menopausal women was negative. Most of the women who expressed a negative attitude toward menopause were below 60 years of age and had limited knowledge related to reproductive health-related knowledge about the natural ageing part of menopause. The women of 60 years old and above understood and accepted menopause as a natural phenomenon in life and part of ageing. This indicates that they accepted menopause more positively than women below the age of 50 years (Robak-Chołubek et al. 2014:666).

The findings of the study provide insight on African women's perception regarding natural menopause. The more aged the African women, the more knowledgeable and experienced about natural menopause they become. This study revealed that 'young' African menopausal women (below the age of 50 years) lack adequate reproductive health-related knowledge about physiological human developmental stages (Robak-Chołubek et al. 2014:666). Inexperience and inadequate health-related knowledge on menopause negatively affects the understanding and perception of natural menopause. African menopausal women need support systems from all relevant stakeholders, including the government, to reduce the cost of curative measures when these women are chronically ill.

# Limitations of the study

The findings of this study were obtained from menopausal women, between the ages 45 and 60 years, in Mamelodi, Tshwane district, therefore they cannot be generalised as these were the perceptions of the women in this particular setting. More studies on women's cultural perceptions on natural menopause need to be done in South Africa to explore and describe their attitude regarding this natural phenomenon.

# Recommendations

The second objective of the study was to propose recommendations for healthcare providers in all healthcare settings to provide reproductive health-related knowledge regarding human developmental stages for all women in Tshwane district. The participants in this study suggested that they need to be furnished with reproductive health-related knowledge during the visit to healthcare settings. Based on the findings of the study, the researcher has recommendations to clinical practice, nursing education and nursing research with regard to natural menopause.

## Clinical practice

Healthcare providers should provide health education on menopause to women who visit the clinic for consultations and follow-up on chronic medications. The researcher further recommends that Gauteng Province government's health department, in collaboration with Tshwane district, provide clinic services for menopausal women, which will be managed by their peers to reduce embarrassment and allow for open, free discussion.

## Health education

Tshwane district should include reproductive health and human development, especially female reproductive development, in health education for all female clients in community health clinics. The health education of African women should also include a memorandum of understanding (MoU) with the Tshwane district and the clinics within its jurisdiction.

## Nursing research

The researcher recommends that further studies be conducted on natural menopause for all African menopausal women. The researchers should collaborate with ageing African women in projects which relate to their health issues in order to promote their cooperation and understanding about health-related matters of the ageing African female population.

# Conclusion

The perceptions of women regarding natural menopause revealed that menopause is viewed according to the attitude of the participants, which is influenced by their cultural beliefs and reproductive health knowledge related to menopause, as well as women's ageing process and development. Some African menopausal women perceived menopause as positive whilst others perceived it as negative, depending on the understanding of the participant respectively. There is a lack of information sharing and limited knowledge about natural menopause because of some cultural sensitivity of reproductive topics and fear of embarrassment. It seems as if the Tshwane district clinics do not provide certain services to women in menopause. These are support systems, such as social networking, and equal opportunities for education in reproductive health and menopause (Thekkedath & Joseph 2009:19). Peer groups for menopausal women in Mamelodi, Tshwane district could be a positive step toward health and wellbeing.

There is a need for community health nurses and other healthcare providers to provide menopausal services such as seminars and health education on menopause to women to reduce the health problems that are related to this phenomenon, by preparing the women in an open, free environment and sharing this natural stage with others. One of the functions of nursing is teaching or educating the public through health education, therefore all nurses should educate the clients at the clinic level as well as during home visits. The South African Nursing Council should ensure that all aspects which are currently overlooked when caring for patients and their families because of changed diet-related life styles which impact on human physiological processes, are somehow revived in the practice of nursing.

## Acknowledgements

The authors would like to thank all the participants for providing and sharing information freely.

## Competing interests

The authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.

## Authors' contributions

G.N.M. (University of Pretoria) conducted the whole study, guided by S.R.R. (University of Pretoria) for literature sources and writing. S.R.R. further drafted the article for G.N.M. to follow as a neophyte author. Literature search and corrections from reviews were addressed by S.R.R. F.M.M (University of Pretoria) provided general, overall support and guidance as an experienced and knowledgeable senior academic, ensuring that the article was completed within timeframe.

# References

[^1]: **How to cite this article:** Rikhotso, S.R., Makuwa, G.N. & Mulaudzi, F.M., 2015, 'The perceptions of African women regarding natural menopause in Mamelodi, Tshwane district', *Curationis* 38(2), Art. \#1531, 7 pages. <http://dx.doi.org/10.4102/curationis.v38i2.1531>


abstract: **Objective** To assess the association between consumption of fried foods and risk of coronary heart disease.
  .
  **Design** Prospective cohort study.
  .
  **Setting** Spanish cohort of the European Prospective Investigation into Cancer and Nutrition.
  .
  **Participants** 40 757 adults aged 29-69 and free of coronary heart disease at baseline (1992-6), followed up until 2004.
  .
  **Main outcome measures** Coronary heart disease events and vital status identified by record linkage with hospital discharge registers, population based registers of myocardial infarction, and mortality registers.
  .
  **Results** During a median follow-up of 11 years, 606 coronary heart disease events and 1135 deaths from all causes occurred. Compared with being in the first (lowest) quarter of fried food consumption, the multivariate hazard ratio of coronary heart disease in the second quarter was 1.15 (95% confidence interval 0.91 to 1.45), in the third quarter was 1.07 (0.83 to 1.38), and in the fourth quarter was 1.08 (0.82 to 1.43; P for trend 0.74). The results did not vary between those who used olive oil for frying and those who used sunflower oil. Likewise, no association was observed between fried food consumption and all cause mortality: multivariate hazard ratio for the highest versus the lowest quarter of fried food consumption was 0.93 (95% confidence interval 0.77 to 1.14; P for trend 0.98).
  .
  **Conclusion** In Spain, a Mediterranean country where olive or sunflower oil is used for frying, the consumption of fried foods was not associated with coronary heart disease or with all cause mortality.
author: Pilar Guallar-Castillón; Fernando Rodríguez-Artalejo; Esther Lopez-Garcia; Luz M León-Muñoz; Pilar Amiano; Eva Ardanaz; Larraitz Arriola; Aurelio Barricarte; Genevieve Buckland; María-Dolores Chirlaque; Miren Dorronsoro; José-María Huerta; Nerea Larrañaga; Pilar Marin; Carmen Martínez; Esther Molina; Carmen Navarro; J Ramón Quirós; Laudina Rodríguez; María José Sanchez; Carlos A González; Conchi Moreno-IribasCorrespondence to: P Guallar-Castillón <email@example.com>
date: 2012
references:
title: Consumption of fried foods and risk of coronary heart disease: Spanish cohort of the European Prospective Investigation into Cancer and Nutrition study

# Introduction

Frying is one of the most commonly used methods for cooking in Western countries. When food is fried its nutritional content changes—the food loses water and takes up fat, increasing its energy density.1 2 Frying modifies both the foods and the frying medium because oils deteriorate during frying, especially when reused, through the processes of oxidation and hydrogenation, leading to a loss of unsaturated fats and an increase in trans fats.1 Thus fried food absorbs degradation products of the frying oil.3 At the same time, frying may also improve palatability by making food crunchy.4 5

Fried foods have been associated with various cardiovascular risk factors in cross sectional studies. In Spain, the Pizarra study of 1226 adults showed that consumption of food fried with reused oils was associated with a higher prevalence of arterial hypertension.6 Furthermore, a cross sectional analysis of the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study concluded that consumption of fried foods was positively associated with general and central obesity.7 Similarly, in the SUN (Seguimiento University of Navarra) cohort in Spain, more frequent consumption of fried foods at baseline was associated with a higher risk of becoming overweight or obese during a six year follow-up.8 Lastly, in another cross sectional study in 2090 Italian adults, consumption of fried food was associated with lower high density lipoprotein cholesterol levels and a larger waist circumference.9

Only a few studies have evaluated the effect of fried foods on risk of cardiovascular disease. A case-control study in Costa Rica found no association between consumption of fried foods and risk of non-fatal acute myocardial infarction.10 Another case-control study, INTERHEART, observed a positive association between fried foods and acute myocardial infarction.11

Therefore, although interest in the associations of dietary patterns and specific cooking techniques with risk of disease is increasing,12 little is known about the health effects of fried foods. We evaluated prospectively the association between consumption of fried foods and the risk of coronary heart disease in the EPIC-Spain cohort.

# Methods

The methods of the EPIC project have been reported elsewhere.7 13 14 15 16 For the present analysis we used the data from the Spanish cohort of EPIC, which included 41 438 healthy adults (15 632 men), aged 29-69. Study participants were recruited between 1992 and 1996 in five Spanish regions. Three of these regions are located in the north (Asturias, Gipuzkoa, and Navarra) and two in the south, including the Mediterranean shore (Granada and Murcia). Selection of these regions aimed to maximise variability in diet because food and nutrient intake has traditionally been different between the north and the south of Spain and between the inner regions and coastal areas. To increase variability in diet, participants were also recruited in urban and rural areas from various educational and social sectors. Study participants included mostly blood donors, civil servants, and the general population. Participation rates varied from 55% to 60% between regions. The exposures of interest were assessed at baseline, and participants were followed up until 31 December 2004. All participants gave written informed consent before enrolment.

## Assessment of food consumption

Using a computerised dietary history questionnaire,17 previously validated in Spain18 19 20 and administered by trained interviewers, we obtained data on usual food consumption for each participant. We used household measures, standard units, and a collection of 35 sets of photographs of simple foods, mixed foods, and drinks to quantify the portion of each food (g/day) consumed. Each interview on dietary history lasted about 40-50 minutes.

Participants were asked about food consumed in a typical week during the previous 12 months. Foods consumed at least twice a month were recorded. The dietary history questionnaire recorded information on up to 662 different foods, including those consumed individually and those consumed as part of 203 recipes and regional dishes. Of these foods, 212 were fried. We calculated energy and nutrient intake using the EPIC food composition table.21

Information on cooking methods was collected as part of the dietary history questionnaire. The same food may be cooked by several methods as part of a single recipe. For example, in the preparation of paella (a typical Spanish recipe), rice is both fried and boiled. Thus, fried food was defined as food for which frying was the only cooking method used. Such food could be deep fried or pan fried and it could be battered, crumbed, or sautéed. To estimate the absorption of fat from frying, we used an absorption coefficient for each fried food.18 We also recorded the type of oil used for dressing, cooking, and frying.

## Assessment of non-dietary variables

Information on demographic variables, educational level, smoking, and physical activity was obtained through interview at recruitment. Physical activity was assessed with a validated questionnaire that included activity at work, at home, and during leisure time.22 Participants were also asked if they had diabetes mellitus, hypercholesterolaemia, hypertension, cancer, or angina, or had experienced myocardial infarction or stroke. In addition they were asked to report their menopausal status and any use of oral contraceptives or hormone replacement therapy. Trained observers used standardised procedures to measure weight, height, and waist circumference. The body mass index was calculated as weight (kg) divided by height squared (m^2^).

## Ascertainment and validation of coronary heart disease

At baseline, prevalent coronary heart disease was self reported. During follow-up, we ascertained incident coronary heart disease with a telephone questionnaire (at three years after recruitment) and through record linkage with three sources of information: hospital discharge databases, population based myocardial infarction registries (available in Murcia, Navarra, and Gipuzkoa), and regional mortality registries and the national mortality database (managed by the National Statistical Institute, Madrid, Spain), which provided information on date and cause of death.

Coronary heart disease was classified according to the *International Classification of Diseases*, ninth revision (ICD-9 codes 410-414) and 10th revision (ICD-10 codes I20-I25). A team of trained doctors and nurses validated coronary heart disease events against hospital records and autopsy reports.

Coronary heart disease events were classified on the basis of symptoms, signs, biomarkers, and findings on electrocardiogram or at autopsy, according to standard criteria.23 A definite coronary heart disease event was defined as one meeting all relevant criteria for a fatal or non-fatal acute myocardial infarction, or angina requiring revascularisation (coronary artery bypass graft or percutaneous transluminal coronary angioplasty). We defined probable and possible acute coronary heart disease events as cases where electrocardiogram findings were non-specific or the biomarkers were equivocal or missing.13

## Statistical analysis

Of the 41 438 participants in the cohort, we excluded the following from the analyses: 193 for having coronary heart disease at baseline; 167 for an implausibly high or low dietary consumption, defined as 3 standard deviations either way from the cohort mean (\<788 kcal/day or \>5710 kcal/day); and 321 for lacking data on important variables such as date of coronary heart disease event (n=12), smoking (n=22), diabetes mellitus (n=71), hypercholesterolaemia (n=197), and hypertension (n=60). Thus the analyses were carried out on 40 757 participants.

We used Cox regression to obtain hazard ratios for coronary heart disease according to sex specific quarters of fried food consumption. The quarter (first) with lowest consumption was used as reference. In the Cox models, age was the underlying time variable, with entry time defined as the participant's age at recruitment and exit time as the age at the coronary heart disease event, death, or 31 December 2004, whichever came first. To reduce violations of the proportional hazards assumption we also stratified models by age at recruitment (five year groups), sex, and centre. We built three models, with progressive adjustment for potential confounders to determine their influence on the results. Model 1 was adjusted for energy intake and model 2 was additionally adjusted for educational level, smoking, physical activity (at work, at home, and during leisure time), diabetes mellitus, hyperlipidaemia, cancer, oral contraceptive use, menopause, hormone replacement therapy, ethanol intake, and consumption of non-fried foods (vegetables, fruits, nuts, dairy products, meat, and fish). Model 3 was additionally adjusted for possible mediators of the study association reported in the literature: body mass index, waist circumference, and hypertension. We categorised continuous variables into quarters, except for age, which was analysed as a continuous variable. The P for linear trend was calculated by modeling the quarters for fried food as a continuous variable. Moreover, we also calculated hazard ratios of coronary heart disease and of all cause mortality for a 100 g increase in fried food consumption analysed as a continuous variable.

New analyses were done after censoring the first two years of follow-up and after excluding those who reported a change in their diet during the previous year. To test whether the study association varied with type of oil used for frying (olive oil versus sunflower oil or other vegetable oils), we used the likelihood ratio test, which compared models with and without interaction terms (products of the quarters for fried food and type of oil). We also used the same procedure to test whether results varied by sex.

Statistical significance was set at P\<0.05. The analyses were carried out with SAS, version 9.1 for Windows. This manuscript follows the recommendations of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) initiative.24

# Results

An average 138 g of fried food was consumed daily, including 14 g of oil used for frying. About 7% of the total amount of food consumed was fried. Fried food consumption ranged from 0-817 g/day for men and 0-657 g/day for women. Sixty two per cent of participants used olive oil for frying and the rest used sunflower oil or other vegetable oils.

Participants with a higher fried food intake were younger, of a higher level of education, more often smokers, and less often sedentary at work (table 1). Moreover, they had a lower prevalence of diabetes mellitus and hyperlipidaemia, a higher energy and ethanol intake, and a lower consumption of non-fried vegetables, fruit, dried fruit and nuts, milk products, and non-fried fish (table 2). They also had a lower frequency of diagnosed hypertension. Among women, the frequency of menopausal status decreased with the consumption of fried food (table 2).

 Consumption of fried foods, sociodemographic variables, and lifestyles of study participants at baseline, by quarters of fried food consumption. Values are means unless stated otherwise

| Characteristics | Quarters of fried food consumption |  |  |  |
|----|----|----|----|----|
|  | 1 (lowest) | 2 | 3 | 4 (highest) |
| **Consumption of fried foods** |  |  |  |  |
| Total amount of fried food consumed (g/day) | 47.0 | 105.7 | 158.4 | 249.6 |
| Fried fish (g/day) | 12.6 | 28.8 | 40.6 | 53.6 |
| Fried meat (g/day) | 8.3 | 21.7 | 36.2 | 59.9 |
| Fried potatoes (g/day) | 9.3 | 21.6 | 33.3 | 57.7 |
| Fried eggs (g/day) | 4.0 | 10.7 | 17.1 | 28.0 |
| **Sociodemographic variables** |  |  |  |  |
| Age (years) | 50.4 | 49.5 | 48.9 | 48.3 |
| Educational level (%): |  |  |  |  |
|  No formal education | 41.9 | 36.4 | 31.7 | 27.6 |
|  Primary education | 31.6 | 35.9 | 40.8 | 46.6 |
|  Secondary education | 12.6 | 14.2 | 16.0 | 16.0 |
|  University education | 13.3 | 12.8 | 10.8 | 9.0 |
| **Lifestyle** |  |  |  |  |
| Smoking (%): |  |  |  |  |
|  Never smoker | 58.3 | 59.3 | 59.5 | 59.7 |
|  Current smoker | 22.5 | 22.5 | 23.7 | 24.2 |
|  Former smoker | 19.2 | 18.2 | 16.8 | 16.1 |
| Physical activity at work (%): |  |  |  |  |
|  Sedentary | 22.8 | 22.1 | 20.4 | 18.8 |
|  Standing | 62.1 | 64.2 | 63.7 | 63.4 |
|  Manual labour | 6.2 | 8.5 | 10.3 | 12.3 |
|  Heavy manual labour | 2.2 | 1.9 | 2.1 | 2.5 |
|  Does not work | 3.8 | 3.3 | 3.6 | 3.1 |
| Physical activity at home (METs h/week) | 68.6 | 69.8 | 70.0 | 68.1 |
| Physical activity in leisure time (METs h/week) | 28.7 | 28.2 | 28.6 | 28.4 |

MET=metabolic equivalents. Range of quarters of fried food consumption in g/day for men: first quarter (0-112), second quarter (113-176), third quarter (177-246), and fourth quarter (247-817), and for women: first quarter (0-59), second quarter (60-103), third quarter (104-154), and fourth quarter (155-657).

 Morbidity, menopausal status, use of hormone treatment, consumption of non-fried food, and potential mediators of study association among study participants at baseline, by quarters of fried food consumption. Values are means unless stated otherwise

| Characteristics | Quarters of fried food consumption |  |  |  |
|----|----|----|----|----|
|  | 1 (lowest) | 2 | 3 | 4 (highest) |
| Diseases diagnosed: |  |  |  |  |
|  Diabetes mellitus (%) | 7.5 | 5.1 | 3.9 | 3.2 |
|  Hyperlipidaemia (%) | 24.8 | 20.5 | 18.5 | 16.7 |
|  Cancer (%) | 1.0 | 0.9 | 0.8 | 0.8 |
| Menopause (%)\* | 42.2 | 36.9 | 33.0 | 29.3 |
| Hormone treatment (%)\*: |  |  |  |  |
|  Oral contraceptives | 24.7 | 26.4 | 26.6 | 27.4 |
|  Hormone substitution therapy | 6.4 | 6.2 | 5.5 | 5.6 |
| Other nutrients and foods consumed: |  |  |  |  |
|  Total energy (kcal/day) | 1920.1 | 2133.0 | 2294.8 | 2566.6 |
|  Ethanol (g/day) | 11.5 | 14.5 | 17.0 | 20.4 |
|  Non-fried vegetables (g/day) | 255.0 | 241.1 | 232.3 | 237.4 |
|  Fruit (g/day) | 336.6 | 322.0 | 315.3 | 311.2 |
|  Dried fruit and nuts (g/day) | 5.0 | 4.6 | 4.2 | 3.9 |
|  Milk products (g/day) | 304.7 | 298.1 | 296.9 | 292.0 |
|  Non-fried meat (g/day) | 93.9 | 98.0 | 97.9 | 96.2 |
|  Non-fried fish (g/day) | 37.2 | 29.6 | 26.0 | 23.8 |
| Mediators of study association: |  |  |  |  |
|  Body mass index (kg/m^2^) | 28.5 | 28.3 | 28.1 | 28.1 |
|  Waist circumference (cm) | 92.8 | 92.0 | 91.4 | 91.4 |
|  Hypertension (%) | 23.7 | 20.9 | 18.4 | 17.4 |

See table 1 for range (g/day) of quarters of fried food consumption for men and for women.

\*Calculated for women.

During a median follow-up of 11 years, 606 definite coronary heart disease events occurred (466 myocardial infarctions and 140 anginas requiring revascularisation). There were also 712 definite, possible, or probable coronary heart disease events and 1135 deaths from all causes.

For definite coronary heart disease events, no association with fried food consumption was observed in analyses adjusting for energy intake, age, sex, and centre (table 3, model 1). The hazard ratio of coronary heart disease for the highest versus the lowest quarter of fried food consumption was 0.94 (95% confidence interval 0.72 to 1.23; P for trend 0.52). Similar results were obtained after additional adjustment for the main confounders (table 3, model 2): 1.11 (0.84 to 1.46; P for trend 0.60). Finally, no association was observed between fried food and incident coronary heart disease after adjusting for possible mediators such as body mass index, waist circumference, and hypertension (table 3, model 3). Compared with the first (lowest) quarter of fried food consumption, the multivariate hazard ratios of coronary heart disease were 1.15 (0.91 to 1.45) in the second quarter, 1.07 (0.83 to 1.38) in the third quarter, and 1.08 (0.82 to 1.43) in the fourth quarter (P for trend 0.74). Lastly, after full adjustment, a 100 g increase in the consumption of fried food did not show an association with risk of coronary heart disease (hazard ratio 1.00, 0.90 to 1.11). The results were similar after censoring the first two years of follow-up and after excluding those who reported a change in their diet during the previous year. The results did not vary between those who used olive oil for frying and those who used sunflower oil or other vegetable oils (P for interaction 0.22), or between the sexes (P for interaction 0.19).

 Association between fried food consumption and incidence of definite coronary heart disease and all cause mortality during 11 years of follow-up in Spanish cohort of European Prospective Investigation into Cancer and Nutrition

| Variables | Quarters of fried food consumption |  |  |  | P for trend | For 100 g increase of fried food |
|----|----|----|----|----|----|----|
|  | 1 (lowest) | 2 | 3 | 4 (highest) |  |  |
| Coronary heart disease: |  |  |  |  |  |  |
|  Total No | 10 188 | 10 190 | 10 190 | 10 189 |  |  |
|  Person years | 109 578 | 110 394 | 110 943 | 111 762 |  |  |
|  No of events | 154 | 163 | 150 | 139 |  |  |
|  Model 1\*: hazard ratios (95% CI) | 1 | 1.05 (0.84 to 1.33) | 0.97 (0.76 to 1.24) | 0.94 (0.72 to 1.23) | 0.52 | 0.95 (0.86 to 1.06) |
|  Model 2†: hazard ratios (95% CI) | 1 | 1.15 (0.91 to 1.46) | 1.08 (0.84 to 1.39) | 1.11 (0.84 to 1.46) | 0.60 | 1.01 (0.91 to 1.12) |
|  Model 3‡: hazard ratios (95% CI) | 1 | 1.15 (0.91 to 1.45) | 1.07 (0.83 to 1.38) | 1.08 (0.82 to 1.43) | 0.74 | 1.00 (0.90 to 1.11) |
| All cause mortality: |  |  |  |  |  |  |
|  Total No | 10 188 | 10 190 | 10 190 | 10 189 |  |  |
|  Person years | 110 135 | 111 019 | 111 560 | 112 342 |  |  |
|  No of deaths | 328 | 276 | 273 | 258 |  |  |
|  Model 1\*: hazard ratios (95% CI) | 1 | 0.90 (0.76 to 1.07) | 0.91 (0.77 to 1.09) | 0.89 (0.73 to 1.08) | 0.26 | 0.98 (0.91 to 1.06) |
|  Model 2†: hazard ratios (95% CI) | 1 | 0.94 (0.79 to 1.11) | 0.95 (0.80 to 1.14) | 0.94 (0.77 to 1.15) | 0.60 | 1.00 (0.92 to 1.08) |
|  Model 3‡: hazard ratios (95% CI) | 1 | 0.94 (0.79 to 1.11) | 0.95 (0.79 to 1.13) | 0.93 (0.77 to 1.14) | 0.98 | 1.00 (0.92 to 1.08) |

Quarters are sex specific. See table 1 for range (g/day) of quarters of fried food consumption for men and for women.

\*Adjusted for age, sex, centre (Asturias, Gipuzkoa, Granada, Murcia, or Navarra), and energy intake (quarters).

†Adjusted for variables in model 1 and for ethanol consumption (quarters among consumers), educational level (no formal education, primary, vocational, secondary, university education), smoking (never, current, former), physical activity at work (sedentary, standing, manual labour, heavy manual labour, no work), physical activity at home (metabolic equivalents h/week in quarters), physical activity in leisure time (metabolic equivalents h/week in quarters), diabetes mellitus, hyperlipidaemia, cancer, oral contraceptives, menopause, hormone replacement therapy, and consumption of fruit (quarters), nuts (quarters), and dairy products (quarters), and non-fried foods (quarters): vegetables, meat, and fish.

‡Adjusted for variables in model 2 and for body mass index (\<25 and ≥25, \<30 and ≥30, \<35 and ≥35), waist circumference (quarters), and hypertension.

Similar results were also obtained in the combined analysis of definite, possible, and probable coronary heart disease events. Compared with the first (lowest) quarter of fried food consumption, the multivariate hazard ratio of coronary heart disease in the fully adjusted model (model 3) was 1.08 (0.88 to 1.34) for the second quarter, 1.06 (0.84 to 1.33) for the third quarter, and 1.04 (0.81 to 1.34) for the fourth quarter (P for trend 0.83).

Fried food consumption also failed to be associated with all cause mortality. Compared with the lowest quarter of fried food consumption, those in the highest quarter had a hazard ratio of 0.93 (0.77 to 1.14; P for trend 0.98). Moreover, no association was observed between a 100 g increase in intake of fried food and total mortality (1.00, 0.92 to 1.08; table 3, model 3).

Of the total amount of fried food consumed, 24% (34 g/day) was fish, 22% (31 g/day) meat, 21% (30 g/day) potatoes, and 11% (15 g/day) eggs. No association was observed between each of these fried food groups and incident coronary heart disease (table 4). Comparing the highest and lowest quarters, the multivariate hazard ratio for the fully adjusted model was 1.13 (0.89 to 1.44; P for trend 0.26) for fried fish, 1.09 (0.82 to 1.43; P for trend 0.32) for fried meat, 0.90 (0.70 to 1.15; P for trend 0.45) for fried potatoes, and 0.87 (0.68 to 1.13; P for trend 0.30) for fried eggs. These fried foods also failed to show an association with all cause mortality (table 4).

 Association between consumption of main types of fried food and incidence of definite coronary heart disease and all cause mortality during 11 years of follow-up in Spanish cohort of European Prospective Investigation into Cancer and Nutrition

| Variables | Quarters of fried food consumption |  |  |  | P for trend | For 100 g increase of fried food |
|----|----|----|----|----|----|----|
|  | 1 (lowest) | 2 | 3 | 4 (highest) |  |  |
| **Coronary heart disease** |  |  |  |  |  |  |
| Fried fish: |  |  |  |  |  |  |
|  No of events | 144 | 136 | 149 | 177 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 0.99 (0.78 to 1.26) | 1.06 (0.83 to 1.35) | 1.13 (0.89 to 1.44) | 0.26 | 1.03 (0.82 to 1.29) |
| Fried meat: |  |  |  |  |  |  |
|  No of events | 137 | 158 | 174 | 137 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 1.17 (0.93 to 1.49) | 1.40 (1.09 to 1.79) | 1.09 (0.82 to 1.43) | 0.32 | 1.05 (0.82 to 1.35) |
| Fried potatoes: |  |  |  |  |  |  |
|  No of events | 188 | 151 | 138 | 129 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 0.92 (0.74 to 1.15) | 0.95 (0.75 to 1.19) | 0.90 (0.70 to 1.15) | 0.45 | 0.91 (0.71 to 1.17) |
| Fried eggs: |  |  |  |  |  |  |
|  No of events | 184 | 151 | 145 | 126 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 0.99 (0.80 to 1.24) | 0.95 (0.76 to 1.20) | 0.87 (0.68 to 1.13) | 0.30 | 0.84 (0.51 to 1.40) |
| **All cause mortality** |  |  |  |  |  |  |
| Fried fish: |  |  |  |  |  |  |
|  No of events | 297 | 276 | 271 | 291 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 0.99 (0.84 to 1.17) | 0.97 (0.81 to 1.15) | 0.92 (0.77 to 1.10) | 0.34 | 0.84 (0.69 to 1.01) |
| Fried meat: |  |  |  |  |  |  |
|  No of events | 346 | 246 | 264 | 279 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 0.80 (0.67 to 0.95) | 0.90 (0.75 to 1.08) | 0.94 (0.78 to 1.14) | 0.72 | 0.96 (0.79 to 1.16) |
| Fried potatoes: |  |  |  |  |  |  |
|  No of events | 353 | 273 | 249 | 260 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 0.90 (0.77 to 1.06) | 0.92 (0.78 to 1.09) | 0.99 (0.83 to 1.18) | 0.87 | 1.10 (0.91 to 1.32) |
| Fried eggs: |  |  |  |  |  |  |
|  No of events | 350 | 265 | 245 | 275 | — | — |
|  Hazard ratio (95% CI)\* | 1 | 0.96 (0.81 to 1.13) | 0.90 (0.75 to 1.06) | 1.05 (0.87 to 1.25) | 0.90 | 1.11 (0.76 to1.64) |

\*Adjusted the same as model 3 in table 3.

Range of quarters of fried food consumption in g/day: fish consumption for men: first quarter (0-15), second quarter (16-35), third quarter (36-61), and fourth quarter (62-439), and for women: first quarter (0-8), second quarter (9-23), third quarter (24-40), and fourth quarter (41-346); meat consumption for men: first quarter (0-12), second quarter (13-34), third quarter (35-65), and fourth quarter (66-384), and for women: first quarter (0-2), second quarter (3-15), third quarter (16-36), and fourth quarter (37-347); potato consumption for men: first quarter (0-12), second quarter (13-33), third quarter (34-59), fourth quarter (60-560), and for women: first quarter (0-3), second quarter (4-17), third quarter (18-34), and fourth quarter (35-282); egg consumption for men: first quarter (0-6), second quarter (7-15), third quarter (16-30), fourth quarter (31-176), and for women: first quarter (0-0), second quarter (1-8), third quarter (9-17), and fourth quarter (18-123).

# Discussion

This analysis of the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition found no association between consumption of fried food and risk of coronary heart disease or all cause mortality.

## Comparison with other studies

Few studies have evaluated this association. Our results are consistent with those of a case-control study of non-fatal myocardial infarction in Costa Rica, which obtained an odds ratio of 1.06 (0.59 to 1.91; P for trend 0.65) for the highest versus lowest fifth of fried food consumption.10 However, in the Costa Rican investigation, palm and partially hydrogenated soy oil were used for frying, whereas in this study mainly olive and sunflower oils—the most commonly used oils in Mediterranean countries—were used.

Our results differ from those of the INTERHEART study, where fried food consumption was associated with a higher risk of acute myocardial infarction.11 However, some methodological differences limit the comparison of results. For instance, INTERHEART did not assess quantitatively the intake of fried food; moreover, it recorded the consumption of only nine fried foods and it did not report the type of oil used for frying.

In a cross sectional analysis of the Multi Ethnic Study of Atherosclerosis (MESA), no association was found between consumption of fried fish and subclinical atherosclerosis, assessed by the thickness of intima-media in the common carotid. Nor was fried fish consumption associated with significant changes in serum levels of total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, or tryglicerides.25 The Cardiovascular Health Study, a cohort of 3919 older people (≥65 years) followed for nine years, also did not find an association of consumption of fried fish or fish sandwiches with mortality from coronary heart disease or cardiac arrhythmia, or with incidence of non-fatal acute myocardial infarction.26

## Possible mechanisms of the study association

The frying process is complex and as yet is not well understood. Laboratory investigations show that fried foods may act through many mechanisms, such that the resulting effect on coronary heart disease is difficult to anticipate. Frying changes the quality and composition of food. For example, frying usually augments the fat content, and for some foods, such as potatoes, the dietary fibre is increased owing to the formation of resistant starch.1 Frying can specifically increase the amount of trans fatty acids in foods.27 The amount depends on the frying technique (deep or pan frying), the degree of thermal degradation of the oil, the type of food, and, above all, the type of oil (the degree of oil unsaturation increases the formation of trans fatty acids).28 However, some foods such as certain types of fish show minimal variations in the content of trans fatty acids after frying, regardless of whether olive oil or sunflower oil is used.29 On the other hand, the use of solid fats for frying can also increase the content of trans fatty acids.

The association between consumption of fried foods and endothelial damage is controversial. In a cross sectional analysis of the Multi Ethnic Study of Atherosclerosis (MESA), consumption of fried fish was inversely associated with the level of soluble intercellular adhesion molecule-1, a marker of endothelial activation. The authors did not expect this finding, since it was not observed for consumption of non-fried fish.30 In contrast, meals rich in oils previously used for deep frying in fast food restaurants have been associated with endothelial damage.31 In another study, however, foods rich in oils previously used for frying and high in oxidation products increased postprandial triglyceridaemia but did not affect endothelial function.32

Other mechanisms may be operating during the frying process. For example, in an in vitro study fried eggs increased the production of angiotensin converting enzyme inhibitory peptides more than boiled eggs did.33 On the other hand, frying also increases the levels of cholesterol oxidation products34 and reduces paraoxonase activity, an enzyme that inhibits oxidation of low density lipoprotein cholesterol.35

This analysis of the EPIC-Spain cohort suggests that the above mentioned mechanisms may offset each other in the general population, with the result that frying with mainly olive oil or sunflower oil is not associated with a higher risk of coronary heart disease. Frying with other types of fats may still be harmful.

## Limitations and strengths of the study

This study has some limitations. Firstly, diet was measured only at baseline. Secondly, some residual confounding cannot be ruled out, since diabetes mellitus, hypertension, and hypercholesterolaemia were self reported. In Spain, however, evidence exists of the diagnostic validity of self reported diabetes mellitus and hypertension.36 37 Thirdly, some degree of under-reporting of fried food consumption is possible, given that people perceive it as unhealthy; and non-differential under-reporting would bias the hazard ratio towards 1, hiding a possible association. Fourthly, we do not know the type of frying procedure (deep or pan) carried out, the extent that oils were reused, and the time and temperature used for frying. It is possible that these factors are differentially associated with coronary heart disease. Fifthly, we cannot separate the effect of a specific food from the effect of the cooking method used. For instance, we cannot separate the effect of frying with oil (for example, olive oil) from the cardioprotective effect of omega 3 fatty acids in fish. Sixthly, results are based on what people ate between 1992 and 1996. Unfortunately, since then no studies have been published on the consumption of fried food by the Spanish population. Data from household consumption surveys, however, indicate that the macronutrient intake has not changed materially from 1991 to 200638; also, overall adherence to the Mediterranean pattern seems fairly stable in the period 1998 to 2005,39 although an increase in the consumption of non-alcoholic beverages and dairy products and a decrease in consumption of potatoes and legumes has been observed over the past two decades.38 Finally, although average consumption of fried food in the highest quarter was fairly high, our results cannot rule out that a fried food intake higher than that observed in this study is associated with coronary heart disease.

This analysis also has important strengths, such as the use of a validated dietary history with quantitative information on the consumption of a large number of fried foods, the large sample size, long follow-up, and adjustment for numerous potential confounders.

## Conclusions

In a Mediterranean country where olive and sunflower oils are the most commonly used fats for frying, and where large amounts of fried foods are consumed both at and away from home, no association was observed between fried food consumption and the risk of coronary heart disease or death.

Our results are directly applicable only to Mediterranean countries with frying methods similar to those in Spain. Firstly, oil (mainly olive and sunflower) rather than solid fat is used for frying in Spain. It is well established that olive oil is less prone to oxidation than other edible oils or fats.40 41 Secondly, consumption of fried foods in Spain is not a proxy for fast food intake. Fast foods are generally prepared by deep frying with oils used several times, and are consumed mostly away from home. In Spain, fried foods are consumed both at and away from home, and both deep frying and pan frying are used. Moreover, we can assume that oil is not reused many times for foods consumed at home; however, the cardiovascular effects of food fried with overly reused oils merit further research. Finally, consumption of fried snacks high in salt is fairly low in Spain, whereas in other countries such as the United States they provide an important percentage of energy intake.42

### What is already known on this topic

1.  The consumption of fried food has been associated with some cardiovascular risk factors, such as hypertension, obesity, and low levels of high density lipoprotein cholesterol

2.  The association between fried food consumption and coronary heart disease has been evaluated in some case-control studies, with heterogeneous results

### What this study adds

1.  In Spain, a Mediterranean country that uses olive or sunflower oil for frying, the consumption of fried foods was not associated with risk of coronary heart disease or all cause mortality

2.  Frying with other types of fats, reusing oils several times, or consuming fried snacks high in salt may still be harmful

Cite this as: *BMJ* 2012;344:e363


date: 2014-01-10
title: Findings of research misconduct.

# Findings of Research Misconduct

**Notice Number:** NOT-OD-14-039

**Key Dates
Release Date:** January 9, 2014

**Related Announcements
** None

None

**Issued by
** Department of Health and Human Services ([DHHS](http://www.hhs.gov/))**
**

Purpose

Notice is hereby given that the Office of Research Integrity (ORI) has taken final action in the following case:

Baoyan Xu, M.D., Ph.D., National Heart, Lung, and Blood Institute, National Institutes of Health: Based on allegations made by readers of a published paper,^[1](#ftn1)^ additional review by the National Institutes of Health (NIH) and ORI, and a limited admission by the Respondent that \`\`some better looking strips were repeatedly used as representatives for several times \[sic\],' ORI found that Dr. Baoyan Xu, formerly a Postdoctoral Fellow, Hematology Branch, Systems Biology Center, National Heart, Lung, and Blood Institute (NHLBI), NIH, and currently at the Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chonqing, China, engaged in research misconduct in research supported by intramural research at NHLBI, NIH.

The questioned research involves a Western blot analysis of IgM and IgG antibodies from Chinese subjects in patients with non-A-E hepatitis and control subjects to test reactivity towards a newly discovered virus. Analysis of Figure 6 of the published paper and Figure S4 of the online supplemental information identified thirteen pairs of Western blot bands which had a common origin yet were labeled as from different subjects and usually as detecting a different class of immunoglobulin. Specifically the following pairs were shown to match using forensically useful tools in Photoshop. Each represent a falsification in one or both of the figures as indicated in the table:

| Identity of Strips | Located in:           |
|--------------------|-----------------------|
| A1 IgM/ F1 IgG     | Fig. 6 & Fig. S4      |
| B6 IgM/E1 IgM      | Fig. 6 & Fig. S4      |
| D7 IgM/A11 IgG     | Fig. 6 & Fig. S4      |
| G3 IgM/H4IgG       | Fig. S4               |
| H9 IgM/F4 IgG      | Fig. S4               |
| A4 IgM/E2 IgG      | Fig. S4               |
| A5 IgM/B9 IgM      | Fig. S4               |
| C9 IgG/C6 IgM      | Fig. S4               |
| D11 IgM/H11 IgG    | Fig. S4               |
| D5 IgM/A1 IgG      | Fig. S4               |
| A10 1GM/F7 IgG     | Fig. S4               |
| C11 1gM/E9 IgG     | Fig. 6                |
| F3 IgG/E9 IgM      | F3 in S4/E9 in Fig. 6 |

The Respondent agreed to correction of Figures 6 and S4 of the PNAS paper.

Dr. Xu has entered into a Voluntary Settlement Agreement and has voluntarily agreed for a period of three (3) years, beginning on December 6, 2013:

\(1\) That prior to the submission of an application for U.S. Public Health Service (PHS) support (including NIH support) for a research project on which the Respondent's participation is proposed, and prior to Respondent's participation in any capacity on PHS-supported research, Respondent shall ensure that a plan for supervision of her duties is submitted to ORI for approval; the supervision plan must be designed to ensure the scientific integrity of Respondent's research contribution; Respondent agrees that she shall not participate in any PHS-supported research until such a supervision plan is submitted to and approved by ORI; Respondent agrees to maintain responsibility for compliance with the agreed-upon supervision plan;

\(2\) That any institution employing her shall submit, in conjunction with each application for PHS funds, or report, manuscript, or abstract involving PHS-supported research in which Respondent is involved, a certification to ORI that the data provided by Respondent are based on actual experiments or are otherwise legitimately derived, that the data procedures, and methodology are accurately reported in the application, report, manuscript, or abstract, and that the text in such submission is her own or properly cites the source of copied language and ideas; and

\(3\) To exclude herself voluntarily from serving in any advisory capacity to PHS including, but not limited to, service on any PHS advisory committee, board, and/or peer review committee, or as a consultant.

^1^Xu, B., Zhi, N., Hu, G., Wan, Z., Zheng, X., Liu, X., Wong, S., Kajigaya, S., Zhao, K., Mao, Q., & Young, N.S. \`\`Hybrid DNA virus in Chinese patients with seronegative hepatitis discovered by deep sequencing.' Proc. Natl. Acad. Sci. (US) 110(25):10264-10269; hereafter referred to as the PNAS paper.'

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abstract: Manganese dioxide nanofibers with length ranged from 0.1 to 1 μm and a diameter of about 4–6 nm were prepared by a chemical precipitation method. Composite electrodes for electrochemical supercapacitors were fabricated by impregnation of the manganese dioxide nanofibers and multiwalled carbon nanotubes (MWCNT) into porous Ni plaque current collectors. Obtained composite electrodes, containing 85% of manganese dioxide and 15 mass% of MWCNT, as a conductive additive, with total mass loading of 7–15 mg cm^−2^, showed a capacitive behavior in 0.5-M Na~2~SO~4~ solutions. The decrease in stirring time during precipitation of the nanofibers resulted in reduced agglomeration and higher specific capacitance (SC). The highest SC of 185 F g^−1^ was obtained at a scan rate of 2 mV s^−1^ for mass loading of 7 mg cm^−2^. The SC decreased with increasing scan rate and increasing electrode mass.
author: Jun Li; QuanMin Yang; Igor Zhitomirsky
date: 2010
institute: 1Department of Materials Science and Engineering, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4L7, Canada; 2Vale Inco Limited, Mississauga, ON, L5K 1Z9, Canada
references:
title: Composite Electrodes for Electrochemical Supercapacitors

# Introduction

Porous Ni materials, such as plaques \[1\] and foams \[2\], are widely used in industry for the fabrication of electrodes for rechargeable batteries. Nanostructured active materials are impregnated chemically or electrochemically into the porous Ni structures, which are used as current collectors \[3\]. Ni plaques are current collectors of choice for battery applications demanding high power and reliability, along with long cycle life, such as batteries for aerospace and railway applications, power tools and some portable electronics \[1\]. The pore size of Ni plaques is smaller compared to that of foams. The smaller pore size decreases the distance for electrons to travel from the current collector into the active material during cell discharge and consequently improves the discharge rate characteristics for high power applications \[3\]. Significant advances in the development of Ni plaques were achieved by the use of filamentary Ni particles with high surface area, which improved contact with active materials \[4\].

A new wave of interest in the application of porous Ni materials is related to the development of electrochemical supercapacitors (ES) \[5\]. ES can complement or replace batteries in electrical energy storage applications when high-power delivery is required \[6\]. Manganese dioxides with various crystalline structures are important materials for electrodes of ES \[7\]. The interest in the application of manganese dioxide in ES is related to low cost of this material, which exhibits high SC in environmentally friendly aqueous electrolytes \[8\].

The charging mechanism of manganese dioxide electrodes of ES is described by the following reaction \[9\]:

![](1556-276X-5-512-i1.gif)

where A^+^ = Li^+^, Na^+^, K^+^, H^+^. Equation 1 indicates that high electronic and ionic conductivity of the electrode material are important in order to utilize the high theoretical capacitance (1,370 F g^−1^) of manganese dioxide \[10\]. Composite \[11\] and thin film electrodes \[12\] were developed and investigated. The properties of MnO~2~ are influenced by crystalline structure, particle size, porosity and surface area \[13\]. A complicating factor in the application of MnO~2~ in ES is low electronic conductivity of this material. This problem can be addressed by the use of advanced current collectors, such as Ni plaques.

In a previous investigation \[14\], manganese dioxide nanofibers were prepared by a chemical precipitation method and utilized for the fabrication of composite manganese dioxide—MWCNT films by electrophoretic deposition. The composite films deposited on stainless steel foils showed high SC in a voltage window of 1.0 V. However, the SC decreased significantly with increasing film thickness. The results presented below indicated that relatively high SC can be achieved at high active material loading using Ni plaques as current collectors. In this approach, the high surface area and porous structure of the Ni plaques provided improved electrical contact of the current collector with active material and enabled good electrolyte access to the active material. We presented experimental results on the fabrication of composite electrodes, investigation into electrode microstructure and electrochemical behavior.

# Experimental Procedures

Manganese dioxide nanofibers were prepared by the method described in the previous investigation \[14\]. In this approach, precipitation was performed by the reduction of 0.2-M KMnO~4~ (Aldrich) solutions with ethanol using the following reaction:

![](1556-276X-5-512-i2.gif)

MWCNT were provided by Arkema company. The average diameter of MWCNT was \~15 nm and length \~0.5 μm. Ni plaques with mass of 0.1 mg cm^−2^ were provided by Inco company and impregnated with a manganese dioxide slurry containing 15 mass% of MWCNT.

XRD studies were performed with a diffractometer (Nicolet I2) using monochromatic Cu Kα radiation at a scanning speed of 0.5 deg min^−1^. TGA and DTA of the manganese dioxide nanofibers were carried out in air at a heating rate of 5 °C/min using a thermoanalyzer (Netzsch STA-409). Electron microscopy investigations were performed using a JEOL 2010F transmission electron microscope and a JEOL JSM-7000F scanning electron microscope equipped with energy-dispersive spectroscopy.

Capacitive behavior of the electrodes was studied using a potentiostat (PARSTAT 2273, Princeton Applied Research) controlled by a computer using a PowerSuite electrochemical software. Electrochemical studies were performed using a standard three-electrode cell containing 0.5 M-Na~2~SO~4~ aqueous solution degassed with purified nitrogen gas. The surface area of the working electrodes was 1 cm^2^. The counter electrode was a platinum gauze, and the reference electrode was a standard calomel electrode (SCE). Cyclic voltammetry (CV) studies were performed within a potential range of 0–1.0 V versus SCE at scan rates of 2–100 mV s^−1^. The SC was calculated using half the integrated area of the CV curve to obtain the charge (*Q*), and subsequently dividing the charge by the mass of the active material (*m*) and the width of the potential window (Δ*V*):

![](1556-276X-5-512-i3.gif)

Impedance spectroscopy investigations were performed in the frequency range of 0.1 Hz–100 kHz at amplitude voltage of 5 mV. Simulations of the impedance behavior were performed on the basis of the equivalent-circuit models using ZsimpWin 3.10 commercial software.

# Results and Discussion

In the previous investigation \[14\], manganese dioxide nanofibers were prepared by the reduction in KMnO~4~ solutions with ethanol, followed by stirring of obtained suspensions during 20 h, and then washing and drying of the suspension. As an extension of the previous investigation, the stirring time was varied in the range of 1–20 h. Figure 1a, 1b show typical TEM images of the nanofibers obtained after 1 h of stirring. The diameter of the nanofibers was 4–6 nm and length 0.1–1 μm. It was found that the nanofibers were not individual crystals but were composed of small nanoparticles with typical size less than 5 nm. The diameter of the nanofibers was non-uniform (Fig. 1b). The increase in the stirring time from 1 to 20 h resulted in partial agglomeration of the nanofibers, which formed bundles (Fig. 1c), containing individual nanofibers. This result was in a good agreement with the results of investigation into other nanomaterials \[15\], which indicated that stirring of nanoparticles can promote agglomeration.

XRD studies (Fig. 2) showed changes in the diffraction patterns of the nanofibers with increasing stirring time. The XRD pattern of the nanofibers stirred for 1 h showed XRD peaks of birnessite, corresponding to the JCPDS file 87-1497. According to the literature, birnessite has a two-dimensional layered structure that consists of edge-shared MnO~6~ octahedra with cation and water molecules occupying the interlayer regions \[16\]. The birnessite formula is generally expressed as A~*x*~MnO~2+*y*~(H~2~O)~*z*~, in which A represents an alkali metal cation. The average oxidation state of Mn usually falls between 3.6 and 3.8, which represents a predominance of Mn^4+^ with minor amounts of Mn^3+^ and Mn^2+^\[16\]. The intensity of the peaks of the birnessite phase decreased with increasing stirring time, indicating lower crystallinity. After 20 h of stirring, the XRD pattern showed very small broad peaks. It was suggested that the nanofibers contained crystalline and amorphous phases.

It was found that the precipitated manganese dioxide nanofibers contained potassium and adsorbed water. EDS studies showed the K/Mn atomic ratio of 0.16 ± 0.03 and 0.14 ± 0.03 for nanofibers stirred during 1 and 20 h, respectively. Figure 3 shows TGA and DTA data for the nanofibers stirred during 1 h. The TGA studies of the powders showed the mass loss related to the dehydration at temperatures below 400 °C. Corresponding DTA data showed a broad endotherm at \~150 °C. Small mass gain in the range of 400–420 °C in TGA data and corresponding DTA exothermic peak can be attributed to oxidation of non-stoichiometric manganese dioxide. The mass loss in the range of 880–900 °C and corresponding endotherm can be attributed to reduction of manganese dioxide in agreement with the literature data \[17\]. Similar behavior was observed for the nanofibers stirred during 20 h.

The slurries containing manganese dioxide nanofibers and MWCNT were used for the impregnation of Ni plaques. Figure 4a shows a schematic of the cross-section of a Ni plaque, which consists of a perforated Ni foil and sintered Ni particles. The voids between the particles provide a space for the loading of the plaques with an active material. Figure 4b, 4c show SEM images of the surface of a Ni plaque at different magnifications. The plaques exhibited porous microstructure with pore size in the range of 1–50 μm (Fig. 4b). The SEM image obtained at higher magnification (Fig. 4c) showed small particle size of Ni particles in the range of 0.5–3 μm, which formed a conductive porous matrix. It was suggested that high surface area, small particle size, porosity and conductivity of Ni plaques are beneficial for application in ES. The SEM image of the impregnated material showed porous fibrous microstructure (Fig. 5) containing manganese dioxide nanofibers and MWCNT.

In the previous investigation \[14\], it was found that manganese dioxide nanofibers and MWCNT formed a porous fibrous network, which was beneficial for the electrolyte access to the active material. However, the SC of the films deposited on metal foil substrates decreased significantly with increasing film mass from 50 to 300 μg cm^−2^. In contrast, the results presented below showed that relatively high SC can be obtained for active material loading of 7–15 mg cm^−2^ using Ni plaque current collectors. In this approach, porous Ni current collectors \[2\] provided improved contact with active material, whereas MWCNT was used as a conductive additive.

Figure 6 shows typical CVs for the composite electrode with material loading of 7 mg cm^−2^. Within the potential range of 0–1.0 V versus SCE, the composite electrode exhibited capacitive-like current-potential responses, indicated by the box shape of the CVs. It is clear from the Fig. 6 that there are no redox peaks in the range between 0 and 1.0 V. Figure 7 shows SC at different scan rates for the composite electrodes. The SC decreased with increasing scan rate due to the diffusion limitations in pores. The electrodes prepared using powders stirred during 1 h showed higher SC compared to the powders stirred during 20 h. This can be attributed to low agglomeration of the powders stirred during 1 h. This result indicated that further optimization of the powder processing conditions can be beneficial for the improvement in electrode performance. The highest SC of 185 F g^−1^ was achieved at a scan rate of 2 mV s^−1^ for material loading of 7 mg cm^−2^. However, the SC decreased with increasing material loading (Fig. 8). The reduction in the SC is especially evident at a scan rate of 100 mV s^−1^. Turning again to the results of the previous investigation \[14\], it is seen that due to the use of Ni plaque current collectors, the material loading can be increased by 1–2 orders of magnitude, and relatively high SC can be obtained. Moreover, the SC (Fig. 8) at a scan rate of 100 mV s^−1^ for the 7 mg cm^−2^ loading of Ni plaque (SC = 111 F g^−1^) was higher compared to the SC of the 0.24 mg cm^−2^ film (SC = 82 F g^−1^) deposited on a foil substrate \[14\]. The high SC at high scan rates is important for the fabrication of efficient ES \[6\].

Figure 9 shows charge–discharge behaviour for the composite electrode with material loading of 10 mg cm^−2^. The charge–discharge curves obtained at different current densities were nearly linear and indicated good capacitive behavior in agreement with box shape CVs shown in Fig. 6. Figure 10 shows impedance spectroscopy data for the sample of the same mass. The equivalent circuit of ES was discussed in the literature and included RC transmission line, describing the porous electrode \[5\]. C~*n*~ elements represent double-layer capacitance and pseudo capacitance, whereas R~*n*~ elements represent electrolyte resistance in pores, Faradaic resistance and equivalent series resistance of the electrodes \[18\]. Conway and Pell described the impedance of porous electrode using 5-element (*n* = 5) circuit \[5\]. A constant phase impedance (CPE) element, rather than a pure capacitance *C*, was used in another investigation \[19\]. The CPE element describes a 'leaking' capacitor with microscopic roughness of the surface and capacitance dispersion of interfacial origin. The equivalent circuits should allow an optimum representation of the measured spectra with a minimum set of model parameters. Good agreement of simulated and measured data (Fig. 10) was found for the equivalent circuit similar to that proposed for composite ruthenium oxide-graphite electrodes \[20\]. In this circuit, *C*~1~ and *R*~1~ described double-layer capacitance and charge transfer resistance, respectively. *Q*~2~ described CPE impedance of porous electrode, and *R*~2~ represented electrolyte resistance. The high frequency *Z*′ value of the complex impedance *Z* = *Z*′ − *iZ*″ showed that *R* = *R*~1~ + *R*~2~ is about 0.4 Ohm for the sample with an area of 1 cm^2^. Relatively low resistance *R* is beneficial for high power ES \[18\].

# Conclusions

Manganese dioxide nanofibers with length ranged from 0.1 to 1 μm and a diameter of about 4–6 nm were prepared by a chemical precipitation method. Composite electrodes for ES, containing two different fibrous materials, were fabricated by impregnation of slurries of the manganese dioxide nanofibers and MWCNT, as a conductive additive, into porous Ni plaque current collectors. The composite electrodes with total mass loading of 7–15 mg cm^−2^ showed good capacitive behavior in the 0.5 M Na~2~SO~4~ solutions. The reduction in stirring time of the precipitated nanofibers resulted in lower agglomeration and higher SC. The highest SC of 185 F g^−1^ was obtained at a scan rate of 2 mV s^−1^ for materials loading of 7 mg cm^−2^. Testing results indicated that Ni plaques are promising current collector materials for application in ES.

## Acknowledgments

The authors gratefully acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada.

### Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


abstract: A report on the American Society for Biochemistry and Molecular Biology symposium 'Transcriptional Regulation by Chromatin and RNA polymerase II', Lake Tahoe, USA, 29 October-1 November 2004.
author: Bing Li; Chun Ruan; Jerry L Workman
date: 2005
institute: 1Stowers Institute for Medical Research, 1,000 East 50th Street, Kansas City, MO 64110, USA; 2The Huck Institute of the Life Sciences, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
title: Histones: should I stay or should I go?

This year's American Society for Biochemistry and Molecular Biology symposium on transcription covered a wide variety of topics ranging from chromatin regulation, through the initiation of transcription and elongation process during transcription by RNA polymerase II (Pol II) to the roles of signaling in transcription and development. This report focuses on the sessions on chromatin, which led to many insightful discussions as a consequence of the rapid advances in this field over the past few years.

# Direct and indirect functions of histone modifications

A central role for chromatin in regulating processes such as transcription and replication is now widely recognized. It is generally believed that the dynamic regulation of chromatin structure makes use of a diverse repertoire of post-translational histone modifications, ATP-dependent chromatin remodeling and histone-variant exchange. Prevailing views on histone modification and its consequences for the modulation of chromatin dynamics, as proposed in the 'histone code hypothesis', suggest that combinations of covalent modifications of specific histone residues comprise a structural and chemical 'code' that can be recognized by other protein modules which then regulate DNA accessibility and function.

Tony Kouzarides (University of Cambridge, UK) presented an interesting case that strongly supports this hypothesis. His group has found that methylation of lysine 20 of histone H4 in the fission yeast *Schizosaccharomyces pombe* is mediated by a novel protein, Set9, that contains a SET domain, a feature that characterizes a subset of chromatin modulators. Unlike other cases of histone lysine methylation, H4 Lys20 methylation appears not to be involved in regulating transcription or the formation of heterochromatin. Instead, it plays a pivotal role in the DNA-damage response pathway. Loss-of-function Set9 mutants and histone H4 K20R (lysine to arginine) mutants prematurely proceed to mitosis by skipping through the checkpoint that can cause arrest between G2 and M phases, even if they have unrepaired DNA. Kouzarides reported that Set9 is required for phosphorylation of the cell-cycle checkpoint protein Crb2, a homolog of the mammalian p53-binding protein 53BP1, and its recruitment to DNA double-strand breaks. He proposed that Crb2 might bind to methylated histone through its Tudor domain, which is present in many RNA-binding proteins. He made the interesting point that the H4 Lys20 methylation itself was not induced upon DNA damage and thus must be present already. Given that Crb2 binds to methylated H4 only at double-strand breaks, it was suggested that high-order structural change in chromatin elicited by DNA damage might be crucial for exposing the buried modified histone tails to their binding partners.

Recent systematic proteomic studies have identified the physical location of a number of novel histone modifications, and Michael Cosgrove (Johns Hopkins School of Medicine, Baltimore, USA) called our attention to certain scenarios in which dynamic chromatin regulation might not be explained by the histone code hypothesis alone. Cosgrove has mapped known histone modifications onto the crystal structure of the nucleosome core particle and he pointed out that a good proportion of them lie in the globular domains of the core histones. In fact, many modifications are positioned at the nucleosome lateral surface, where they would be likely to affect histone-DNA interactions. From a structural perspective he suggested that these modifications might have direct roles in fine tuning histone-DNA contacts. Moreover, for these modifications to be made to pre-assembled nucleosomes, a dramatic conformational change would be required to make specific residues accessible to the modifying enzyme.

Cosgrove proposed a 'regulated nucleosome mobility' model, in which the mobility of nucleosomes is dictated by the affinity of histone octamers for DNA, which is in turn highly regulated by the concerted action of ATP-dependent remodeling factors and histone-modifying enzymes. In this model, remodeling and histone modification can mutually influence each other; this differs from the predictions of the histone-code hypothesis that histone modifications are established first and then direct later remodeling events. The eventual changes in nucleosome mobility can result from either remodeling or covalent modifications or combinations of both. Cosgrove cited genetic evidence to support this argument, noting that some *Sin* (Swi-independent) mutations in histones, which can bypass the requirements for Swi/Snf chromatin-remodeling enzymes or the histone acetyl transferase Gcn5 for transcription, coincide with several modified residues located at histone-DNA contact regions, suggesting that it is nucleosome mobility that matters.

# Nucleosome displacement in transcription

Several presentations at the meeting shed new light on the relationship between nucleosome occupancy and transcriptional activity. In a poster presentation, Cheol-Koo Lee of Jason Lieb's laboratory (University of North Carolina, Chapel Hill, USA) described their work on determining relative nucleosome occupancy throughout the *Saccharomyces cerevisiae* genome. Using chromatin immunoprecipitation (ChIP) assays in combination with DNA microarrays that cover the entire yeast genome at a 1 kilobase (kb) resolution, they found that immunoprecipitating Myc-tagged histone H4 and histone H3 preferentially pulls down more coding DNA than non-coding DNA. This suggests that nucleosomes are depleted from active regulatory elements. By comparing their data to transcription-profile datasets, Lee and colleagues found that the extent of nucleosome occupancy at gene promoters is inversely proportional to the rate of gene transcription. Moreover, when growth conditions are altered, nucleosome occupancy on promoters undergoes some dramatic changes as gene-expression patterns change, but the same inverse correlation still applies. Interestingly, this universal pattern of nucleosome distribution does not appear to rely on specific histone tails, as Lee and colleagues observed similar results in yeast strains bearing various tailless histone mutations.

Kevin Struhl (Harvard Medical School, Boston, USA) reached a similar conclusion - that there is an inverse correlation between nucleosome occupancy and transcription - for the yeast *GAL1* promoter. He also reported that heat inactivation of the Pol II carboxy-terminal domain kinase Kin28 caused a reduction of Pol II occupancy at promoters and coding regions, and a corresponding increase in H3 occupancy of the same regions. This suggests a tight link between the presence of Pol II and histone loss. Using an artificial gene containing a *GAL1* promoter driving a long coding sequence, Struhl and colleagues were able to monitor the last wave of Pol II traveling along the gene after inactivation of the promoter. They discovered that as Pol II transcribed through the gene, histones were immediately deposited onto the DNA behind the polymerase, thus restoring the chromatin structure.

Struhl also presented biochemical data suggesting that promoter DNA may resist nucleosome formation. He and his colleagues reconstituted nucleosomes with yeast genomic DNA *in vitro*, isolated the free DNA from the assembled nucleosomes, and subsequently hybridized it to DNA microarrays of the whole yeast genome. They found that 75-80% of promoter regions were half as likely to reconstitute nucleosomes than were coding regions. This suggests that intrinsic properties of promoter DNA sequence may contribute to nucleosomal distribution in a manner independent of transcription.

David Gross (Louisiana State University Health Sciences Center, New Orleans, USA) has also observed apparent histone eviction in yet another model system. Heat-shock activation of the *S. cerevisiae* *HSP82* gene coincided with loss of nucleosomes from its promoter and coding regions. He claimed that dissociation of histones at *HSP82* was not caused by the global effects of heat shock, as two genes (*YAR1*, *CIN2*) adjacent to *HSP82* did not lose histones. Unlike the scenarios described by Lee, Gross observed a transient hyperacetylation of H2A, H3 and H4 tails at promoter regions prior to the loss of histones; the functional consequence of this is still unclear, however. Most importantly, in this case transcription *per se* does not seem to be the direct cause of these dramatic changes in the chromatin. Deletion of the TATA box reduced *HSP82* transcription to a much lower level than that observed with a promoter mutation that weakens binding of the transcriptional activator heat-shock factor (HSF). Nevertheless, the domain-wide histone eviction is almost unaffected in the TATA mutant but is abolished when HSF can no longer efficiently bind to the upstream sequence. This is in marked contrast to Struhl's results, where transcription seems to have an active role. What causes histone dissociation at *HSP82* is largely unknown; dissociation does, however, seem to be independent of some prominent chromatin remodelers such as Swi/Snf, Gcn5, the histone methylase Set1 and elongation factor Paf1.

From studies to determine which factors might be directly responsible for nucleosome loss, Melissa Adkins of Jessica Tyler's lab (University of Colorado Health Sciences Center, Denver, USA) provided evidence that the histone H3/H4 chaperone Asf1 mediates the loss of nucleosomes at the *PHO5* and *PHO8* promoters upon activation and that histone loss was essential for transcriptional activation of these two yeast genes. This observation implies that in this case histone eviction might not be driven by the Pol II machinery traveling along the DNA; rather, it might be a prerequisite for Pol II transcription to occur after the activator (Pho4) has bound. Adkins also reported that nucleosomes were reassembled onto the *PHO5* promoter during gene repression and that the protein Spt6 might be involved in this reassembly. This is consistent with the known role of Spt6 in repressing faulty transcription from cryptic start sites in coding regions. Unsurprisingly, however, Asf1 cannot be the single answer for all genes because it seems to be unnecessary for the activation of *GAL1* and *HSP82*.

The apparent discrepancies among these reports may very well reflect the diversity and subtlety of chromatin regulation at different genes, or we may simply be missing some important links in our understanding. It is clear, however, that great strides have been made in establishing the paradigm that assembly and disassembly of nucleosomes occurs in a very dynamic manner even within a single cell cycle.