Structural characterization and biological implications of sulfated N-glycans in a serine protease from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae).

Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.

SINGLE AND AGGREGATE SALIVARY CORTISOL MEASURES IN WORKING WOMEN LIVING IN HIGH AND LOW STATUS NEIGHBORHOODS IN SWEDEN.

Contextual factors including neighborhood status have consistently been associated with health disparities. This may relate to a poorer neighborhood status involving an exposure to chronic stressors, which dysregulates cortisol secretion. This study investigated single and aggregate cortisol measures in 88 working women living in high and low status neighborhoods. Results showed significantly lower waking cortisol among women in low status neighborhoods. However, there were no group differences in aggregate cortisol measures. The lower morning cortisol among women in the low status neighborhoods follows previous research suggesting hypocortisolism as a pathway linking neighborhood status and health disparities, albeit a less consistent finding across cortisol measures in this sample. This may relate to the Swedish welfare state and its fostering of equality.

Trapezius activity of fibromyalgia patients is enhanced in stressful situations, but is similar to healthy controls in a quiet naturalistic setting: a case-control study.

BACKGROUND: Muscle activity and pain development of fibromyalgia (FM) patients in response to mental stress show inconsistent results, when compared to healthy controls (HCs). A possible reason for the inconsistent results is the large variation in stress exposures in different studies. This study compares muscle responses of FM patients and HCs for different modes and levels of imposed stress, to elucidate features in stress exposures that distinguish stress responses of FM patients from HCs. METHODS: Upper trapezius (clavicular and acromial fibers), deltoid, and biceps surface electromyographic (sEMG) activity was recorded in FM patients (n=26) and HCs (n=25). Heart rate (HR) was recorded and used as indicator of autonomic activation. Tests included inspiratory breath holding (sympathetic activation procedure), mental stress tests (color-word test and backward counting; 28 min), instructed rest prior to stress test (30 min TV watching), and controlled arm movement. sEMG and HR was also recorded during an unrestrained evening stay at a patient hotel. The 5-min period with lowest trapezius muscle activity was determined. Pain (shoulder/neck, low back pain) and perceived tension were scored on VAS scales at the start and the end of the stress test and at bedtime. RESULTS: Trapezius sEMG responses of FM patients were significantly higher than HCs during sympathetic activation, mental stress, and instructed rest, but similar during arm movement and unrestrained evening activity. HR of FM patients and HCs was similar during mental stress and in the evening, including the 5-min period with lowest trapezius activity. Muscle activity of FM patients during the stress test (with shoulder/neck pain development) and the evening stay (no pain development) was similar. CONCLUSIONS: FM patients show elevated muscle activity (in particular trapezius activity) in situations with imposed stress, including sympathetic activation, and putative anticipatory stress. Muscle activity and HR were similar to HCs in instructed arm movement and in a situation approaching low-stress daily living. Pain development of FM patients during the stress test may be due to activation of several stress-associated physiological systems, and not obviously caused by muscle activity in isolation.

Postpartum depressive symptoms and the combined load of paid and unpaid work: a longitudinal analysis.

PURPOSE: To investigate the effects of total workload and other work-related factors on postpartum depression in the first 6 months after childbirth, utilizing a hybrid model of health and workforce participation. METHODS: We utilized data from the Maternal Postpartum Health Study collected in 2001 from a prospective cohort of 817 employed women who delivered in three community hospitals in Minnesota. Interviewers collected data at enrollment and 5 weeks, 11 weeks, and 6 months after childbirth. The Edinburgh Postnatal Depression Scale measured postpartum depression. Independent variables included total workload (paid and unpaid work), job flexibility, supervisor and coworker support, available social support, job satisfaction, infant sleep problems, infant irritable temperament, and breastfeeding. RESULTS: Total average daily workload increased from 14.4 h (6.8 h of paid work; 7.1% working at 5 weeks postpartum) to 15.0 h (7.9 h of paid work; 87% working at 6 months postpartum) over the 6 months. Fixed effects regression analyses showed worse depression scores were associated with higher total workload, lower job flexibility, lower social support, an infant with sleep problems, and breastfeeding. CONCLUSIONS: Working mothers of reproductive years may find the study results valuable as they consider merging their work and parenting roles after childbirth. Future studies should examine the specific mechanisms through which total workload affects postpartum depressive symptoms.

Concentrations of Salivary Cortisol in Victims of Intimate Partner Violence According to the CIRCORT Database.

This work analyzes the different levels of salivary cortisol in women from the southwest of Spain that were victims of intimate partner violence (IPV) with respect to a control group, assessing for the first time the different concentrations obtained in relation to a worldwide reference standard provided by the CIRCORT meta-global cortisol database. The clinical sample (N = 24) and the control group (N = 25) had an average of 39.12 years (SD = 12.31) and 39.52 years (SD = 11.74), respectively. Cortisol awakening response (CAR) was determined by defining the area under the curve (AUCi). There were no differences between the CAR data of the two populations F (1, 141) = 1.690, p = 0.196, but there was a highly significant difference in the three sampling days, where the clinical sample exceeded the cortisol levels of the CIRCORT database in the evening as compared to the control group (p = 0.004, p = 0.001 and p = 0.000). Salivary cortisol concentration samples taken in the evening were significantly higher than those standardized in the CIRCORT database, from the women victims of IPV as compared to the control group, showing its usefulness as an effective supportive tool for problems such as those triggered by IPV.

Fibromyalgia syndrome is associated with hypocortisolism.

BACKGROUND: Fibromyalgia syndrome (FMS) is a disease of unknown pathogenesis characterized by chronic musculoskeletal pain. FMS has been also associated with altered endocrinological responses, but findings are inconsistent. PURPOSE: The aim of the present study was to investigate free salivary cortisol levels in FMS patients compared with healthy controls with a particular focus on the cortisol awakening response (CAR). The saliva samples were collected in a controlled hospital-hotel setting, in which the participants' compliance was high and a number of potential confounders were analyzed. METHOD: Twenty-nine chronic female FMS patients and 29 age-matched healthy female controls were recruited. Salivary cortisol samples were investigated eight times: in the afternoon when participants arrived at the hospital, after stress provocation (to be reported separately), in the evening, before they went to sleep, upon awakening, 30 and 60 min later, and during the afternoon of the second day. Questionnaires measuring pain levels, sleeping problems, perceived stress, and personality were administered to the participants. Other psychophysiological measurements were used to assess sleep quality and heart rate. RESULTS: Patients with FMS had significantly lower cortisol levels during the day, most pronounced in the morning (CAR). The potential confounders analyzed did not influence the results. As expected, FMS patients reported more pain, stress, sleeping problems, anxiety, and depression. CONCLUSION: The results lend support to the hypothesis of a dysfunction in the hypothalamus-pituitary-adrenal axis in FMS patients, with generally lower cortisol values, most pronounced upon awakening (CAR).

Health-related effects of worksite interventions involving physical exercise and reduced workhours.

OBJECTIVES: This study examined the health-related effects of two worksite interventions, physical exercise and reduced workhours, on women employed in dentistry. METHODS: Six workplaces were randomized to one of the following three conditions: (i) 2.5 hours of weekly, mandatory physical exercise of middle-to-high intensity to be performed during workhours (N=62), (ii) a reduction of full-time weekly workhours from 40 to 37.5 hours (N=50), and (iii) reference. In all, 177 women participated. Biomarkers and self-ratings in questionnaires were obtained before the intervention (T (1)), and six (T (2)) and 12 months (T (3)) after the intervention. RESULTS: The results showed increased levels of physical activity and exercise in all of the groups, the level of physical exercise being significantly greater in the physical exercise group. Repeated-measures analyses of variance using data from T (1)and T (3)for biological measures and all three time points for self-ratings produced significant interaction effects for glucose, waist-to-hip ratio, and work ability and clear trends for general symptoms and upper-extremity disorders. Posthoc analyses showed that the results of the health-related measures differed between the interventions, decreased glucose and upper-extremity disorders in the exercise group, and increased high-density lipoprotein and waist-to-hip ratio among those working reduced hours. CONCLUSIONS: These results show that the two interventions had small and varied effects on biomarkers and self-reports of different aspects of health among women. It is suggested that interventions involving a modest reduction in workhours seem to be more effective if these hours are used for physical exercise.

Mothers' health and work-related factors at 11 weeks postpartum.

PURPOSE: Many new mothers return to work soon after childbirth. This study examines personal and work-related factors associated with the postpartum health of employed women 11 weeks after childbirth. METHODS: Using a prospective cohort design, we recruited 817 Minnesota mothers into the study while they were hospitalized for childbirth in 2001. Telephone interviews were conducted at 5 and 11 weeks postpartum. Eligible women were 18 years or older, employed, and spoke English and gave birth to a singleton infant. Multivariate models using instrumental variables (2-stage least squares) were used to estimate personal and employment characteristics associated with women's physical and mental health and postpartum symptoms. RESULTS: At 11 weeks postpartum, 661 participants (81% of enrollees) completed a full interview, and 50% of participants had returned to work. On average, women reported 4.1 (SD 3.2) childbirth-related symptoms, most frequently fatigue (43%). Factors significantly associated with better health outcomes included better preconception health, the absence of prenatal mood problems, more control over work and home activities, more social support at work and home, and less job stress. CONCLUSIONS: The findings suggest postpartum women need to be evaluated regarding their fatigue levels and mental and physical symptoms. Women whose fatigue or postpartum symptoms limit daily role function may find it helpful to have health care clinicians counsel them on strategies to decrease job stress, increase social support at work and home, and certify their use of intermittent family and medical leave to help them manage their symptoms.

Isolation and partial characterization of proteins with vasodegenerative and proinflammatory properties from the egg-nests of Hylesia metabus (Lepidoptera: Saturniidae).

The Hylesia genus comprises a group of Neotropical moths ubiquitous in the Americas from Arizona to Argentina. One of the species of the Hylesia genus in Venezuela, French Guyana, and Trinidad has been identified as Hylesia metabus (Cramer 1775) (Lepidoptera: Saturniidae). In Venezuela, these moths are found in abundance in the mangrove (Avicennia spp.) swamps surrounding the Gulf of Paria and the Orinoco Delta in the eastern part of the country. During the mating season, the female adults shed copious amounts of urticating setae in the air, producing a severe papulovesicular dermatitis among the population in the affected areas. The females also use their urticating setae to protect the eggs during the hatching period. In the current study, we have isolated and partially characterized proteins with proinflammatory properties from the urticating setae in the egg-nests by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anionic exchange-high-performance liquid chromatography (HPLC). We also have studied the biological response of the egg-nest extract and the HPLC purified fractions by inoculation in guinea pigs; and, analyzing the tissue samples by means of histopathological methods. The results of this study show that the extracted venom and HPLC purified subcomponents give rise to an intense inflammatory reaction characterized by massive infiltration of inflammatory cells, echymoses, and vascular degeneration. Chromatographic separation showed that the venom was made up of proteins having selectively vasodegenerative-fibrinolytic or proinflammatory-quimotactic properties.

Protein content of the Hylesia metabus egg nest setae (Cramer [1775]) (Lepidoptera: Saturniidae) and its association with the parental investment for the reproductive success and lepidopterism.

Hylesia metabus is a neotropical moth possessing toxic setae, which once in contact with the skin cause a severe dermatitis to humans known as lepidopterism. The only known function of the setae in the life cycle is to provide protection during the mating and egg-hatching stages. Approximately 65% of the protein content of the setae is a cluster of five proteases (28-45kDa) showing sequence homology to other S1A serine proteases. The N-glycans of a 40kDa protease are a mixture of neutral and sulfated G0F structures. The sulfated N-glycans have an important role in triggering the inflammatory response typical of lepidopterism while the proteolytic activity may promote the erosion of blood vessels and tissues causing focal hemorrhages. The presence of Chitinase and a 30kDa lipoprotein is probably related to the antifungal defense. In addition, chitin digestion of the setae may potentiate the inflammatory reaction caused by the toxins due to the formation of chitin adjuvants fragments. The combined effect of proteases and a chitinase may dissuade predating arthropods, by damaging their exoskeletons. Vitellogenin, a bacteriostatic protein, is able to recognize pathogen-associated patterns, which suggests its possible role in protecting the embryonated eggs from pathogenic microorganisms. SIGNIFICANCE: The present study is the first report describing the different protein species present in the urticating egg nest setae of the neotropical moth Hylesia metabus - the most harmful of the Hylesia moths - causing a severe urticating dermatitis in humans known as lepidopterism. A distinctive feature of the venom is the presence of five different S1A serine proteases probably used to guarantee a more efficient degradation of a wider number of protein substrates. This work confirms that the presence of sulfated N-glycans is not an isolated finding since its presence has been demonstrated in two different proteases affirming that this PTM is of importance for the activation of the inflammatory response typical of lepidopterism. Additionally, this study gives useful information on the defense mechanisms used for protection of its progeny vs. vertebrate predators, fungus, bacteria or other arthropods such as ants. The proteins detected in the egg nest should be seen as an extended parental effort made by the females in order to achieve an optimal reproductive success, thus compensating for the considerable loss of progeny during the larval stages that seriously limits the number of sexually mature adults reaching the reproductive phase.

Allostatic load and clinical risk as related to sense of coherence in middle-aged women.

OBJECTIVE: To investigate how physiologic dysregulation, in terms of allostatic load and clinical risk, respectively, relates to sense of coherence (SOC) in women with no previously diagnosed pathology. METHODS: At baseline, 200 43-year-old women took part in a standardized medical health examination and completed a 3-item measure of SOC, which they completed again 6 years later. According to data from the medical examination, two different measures of physiologic dysregulation were calculated: a) a measure of allostatic load based on empirically derived cut points and b) a measure of clinical risk based on clinically significant cut points. RESULTS: In line with the initial hypotheses, allostatic load was found to predict future SOC, whereas clinical risk did not. In addition to baseline SOC and nicotine consumption, allostatic load was strongly associated with a weak SOC at the follow-up. CONCLUSIONS: The better predictive value of allostatic load to clinical risk indicates that focusing solely on clinical risk obscures patterns of physiologic dysregulation that influence future SOC.

Postpartum health of employed mothers 5 weeks after childbirth.

PURPOSE: Most new mothers return to work soon after childbirth. A need exists to reexamine the definition of postpartum health and evaluate employed women's recovery from childbirth in association with such factors as delivery type and breastfeeding. METHODS: Using a prospective cohort design, we recruited Minnesota women into the study while they were hospitalized for childbirth in 2001. Telephone interviews were conducted 5 weeks postpartum. Eligible women were 18 years or older, employed, and spoke English. Multivariate models using 2-stage least squares were used to estimate factors associated with physical and mental health and postpartum symptoms. RESULTS: A total of 817 women were enrolled (71% response) in the study; 716 women completed interviews at 5 weeks postpartum. On average, women reported 6 postpartum symptoms, most frequently fatigue (64%), breast discomfort (60%), and decreased desire for sex (52%). Findings showed that cesarean (vs vaginal) deliveries were associated with significantly worse physical function, role limitations, and vitality. Multivariate findings showed that the effect of delivery type on physical health was moderately large (beta = -5.96; P = or <.01), and breastfeeding was associated with an increased frequency of postpartum symptoms (beta = 4.63; P = .01). CONCLUSIONS: These mothers experienced several childbirth-related symptoms at 5 weeks postpartum, indicating a need for ongoing rest and recovery. Health concerns were greater for women who were breastfeeding and for those whose babies were delivered by cesarean section, suggesting a need for greater support for these women and a reassessment by the medical community of the progressively growing practice of cesarean deliveries.

Self-rated recovery from work stress and allostatic load in women.

OBJECTIVE: The objective of this study was to investigate the relationships between self-rated recovery from work stress and biologic dysregulation in terms of allostatic load (AL) and individual biomarkers, respectively, in healthy women within the public health care sector. METHODS: Two hundred forty-one women completed self-ratings of recovery and took part in a standardized medical examination, which provided individual biomarkers that were used to compute AL. RESULTS: Cluster analysis of self-rated recovery resulted in three cluster profiles, including (1) recovered women (n=108), (2) nonrecovered women (n=51), and (3) fatigued women (n=82). Sequential logistic regression analysis showed that the fatigued profile had an increased risk for high AL. In contrast, there was no significant difference in individual biomarkers between recovery profiles. CONCLUSIONS: The findings establish an association between biologic processes and self-rated recovery from work stress, thus supporting the hypothesis that insufficient recovery may result in high AL.

Work characteristics and upper extremity disorders in female dental health workers.

Many dental health workers suffer from musculoskeletal disorders in the upper extremities. In addition to ergonomic factors, psychosocial work characteristics have been linked to musculoskeletal disorders. The present cross-sectional study aimed at investigating how musculoskeletal disorders in the upper extremities (UED) and occupational position are related to work characteristics and general health problems in female dental health workers. Questionnaire data from dentists, dental hygienists and dental nurses (N=945) showed that 81% reported UED. Multivariate analysis of variance showed that dentists reported the highest levels of physical load and fatigue whereas dental nurses reported the lowest levels of influence at work. Irrespective of position, those with UED considered their physical and psychosocial work environment and their own health to be significantly poorer than did those without UED. A hierarchical multiple regression showed that the physical load of dentistry was most strongly related to UED. Despite improvements to the ergonomics and physical work environment of dentistry, it is concluded that female dental health workers are still at high risk of developing UED.

[Changes in serum lipids, plasma fibrinogen and other haemostatic parameters induced by ciprofibrate action in hyperlipidemic patients with and without coronary artery disease]. / Modificaciones de los lipidos séricos, el fibrinógeno y otros parámetros hemostáticos por acción del ciprofibrato, en pacientes hiperlipidémicos con y sin enfermedad arterial coronaria.

The effects of drugs with hypolipidemic properties in the prevention of the atherothrombotic vascular disease, go further than reducing serum lipids, suggesting that there are other nonlipid-related mechanisms involved; the maintenance of appropriate haemostatic balance being one of them. The objective of this investigation was a drug intervention with ciprofibrat in hyperlipidemic people with high level of plasmatic fibrinogen with the purpose of knowing the effects of the drug over these risk factors and other haemostatic parameters. Forty people, both sexes, 20 of them apparently healthy and the other 20 with clinical and angiographic evidence of coronary artery disease, were randomized to receive 100 mg of ciprofibrat or placebo during an average of 56 weeks. All of them had a clinical exam, EKG and stress test. Laboratory exams included lipid profile, plasma fibrinogen (Fg), VII factor, vonWillebrand factor, protein C (PC) and the tissue plasminogen activator with samples taken every 8 weeks. The Ciprofibrat group showed significant changes of lipids: cholesterol -23%, triglycerides -31%, high-density lipoprotein (HDLc) +24% and very low-density lipoprotein -23%, except low-density lipoprotein -24%. The haemostatic parameters in 40 weeks showed that Fg decreased 21% (p = 0.001), decreasing to 9% at the end of the follow-up. In the placebo group the HDLc showed a 10% increase (p = 0.02), PC reduced to 20% (p = 0.01) and Fg kept blood levels close to basal line, descending 10% at the end of the follow-up. In this study, the use of ciprofibrat in patients with high risk of developing atherothrombotic events, showed efficiency and security in handling hyperlipidemia, such as keeping and appropriate haemostatic balance.

Visuospatial and verbal memory in chronic pain patients: an explorative study.

Cognitive bias, such as selective memory for pain-related information, is frequently observed in chronic pain patients and is assessed mostly using verbal material. Beside word lists, the current study used photographs of people presenting pain behaviors to assess memory bias in chronic pain patients. Chronic pain patients were hypothesized to show better recall of pain-related words and pictures as compared to pain-free controls. Twenty-eight female chronic neck patients and 28 pain-free female controls completed two computerized pictorial memory games and two word recall tasks. Patients and controls performed equally well in the neutral pictorial memory game. In the pain-related game, patients performed significantly worse than controls. No significant differences were found in the word recall task. The result is discussed in terms of cognitive avoidance.

Stress hormones in health and illness: the roles of work and gender.

Two neuroendocrine systems are of specific interest in the study of stress and health; the sympathetic adrenomedullary system with the secretion of epinephrine and norepinephrine, and the hypothalamic pituitary adrenocortical (HPA) system with the secretion of cortisol. These hormones have often been used as objective indicators of stress in the individual. However, through their bodily effects, they are also a link between the psychosocial environment and various health outcomes. From a series of studies of women and men, it was concluded that gender roles and psychological factors are more important than biological factors for the sex differences in stress responses. The stress responses have been important for human and animal survival and for protection of the body. However, in modern society, some of these bodily responses may cause harm rather than protection. The catecholamines have been linked to cardiovascular disorders such as hypertension, myocardial infarction and stroke, cortisol to cardiovascular disease, Type 2 diabetes, reduced immune function and cognitive impairment. An adequate balance between catabolic (mobilization of energy) and anabolic processes (growth, healing) is considered necessary for long term health and survival. In modern society, which is characterized by a rapid pace of life, high demands, efficiency and competitiveness in a global economy, it is likely that lack of rest, recovery and restitution is a greater health problem than the absolute level of stress.

Fear, pain and stress hormones during childbirth.

AIMS: To investigate the course of fear, pain and stress hormones during labor, and the associations between fear, pain, stress hormones and duration of labor in nulliparous women with and without epidural analgesia (EDA). METHOD: One day during gestation weeks 37-39, urinary and salivary samples were collected to measure catecholamines and cortisol. Hourly during labor, the participants answered the Delivery Fear Scale and a pain intensity scale, and urinary and salivary samples were collected to measure stress hormones. RESULTS: The course of fear, pain and stress hormones differed throughout labor in women with and without EDA. Pain and cortisol increased throughout labor in women without EDA. Women who received EDA had more fear, but not more pain, before the administration of the EDA than women who did not receive EDA. Pain, fear and catecholamines decreased when women received EDA, but fear and pain increased again later in labor. Fear and pain correlated, as well as levels of fear in the different phases of labor. During phase one of labor epinephrine and duration of the phase were negatively correlated. CONCLUSION: The course of fear, pain and concentrations of stress hormones differed, highly influenced by the administration of EDA. Fear and pain correlated more pronounced than stress hormones and fear, pain and duration of labor.

[Relation of socioeconomic levels and life style to fibrinogen and von Willebrand factor in healthy Venezuelans and those with ischemic cardiopathy]. / Relación entre el nivel socioeconómico y hábitos de vida, con el fibrinógeno y el factor von Willebrand en venezolanos sanos y con cardiopatía isquémica.

Previous studies in Europe, U.S.A and Japan have revealed an inverse relationship between socioeconomic levels and fibrinogen concentration. Similar results have been reported in a smaller number of studies for concentrations of von Willebrand factor. In this opportunity we present results on the relationship between smoking, drinking, physical activity, age and socioeconomic level on fibrinogen and von Willebrand factor concentrations in a Venezuelan sample. The control population consisted of 978 men and 968 women. Patients with coronary heart disease were 172 males and 78 females. The presence of one or more of the following conditions: smoking or less than 5 years of having quit, non drinkers or drinking in excess, and a reduced physical activity, was considered a health related risk factor for high levels of these two haemostatic variables. Our results indicate that in Controls, the socioeconomic level had a significant effect on fibrinogen and von Willebrand factor levels, only in women: those of lower socioeconomic levels had the highest concentrations. This difference was maintained when age was taken into account. Health related behaviors had no significant effect on either variable. In patients, age had no effect on either variable. The health behavior risk factor had a significant effect only on fibrinogen of male patients, and socioeconomic level had a significant effect only on the fibrinogen of female patients. More studies in Venezuela are recommended, in order to increase our knowledge on the relationship between socioeconomic levels, haemostatic markers and the occurrence of coronary heart disease.