Highly heterogeneous nature of delta-aminolevulinate dehydratase (ALAD) deficiencies in ALAD porphyria.

The properties of 9 delta-aminolevulinate dehydratase (ALAD) mutants from patients with ALAD porphyria (ADP) were examined by bacterial expression of their complementary DNAs and by enzymologic and immunologic assays. ALADs were expressed as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and purified by glutathione-affinity column chromatography. The GST-ALAD fusion proteins were recognized by anti-ALAD antibodies and were enzymatically active as ALAD. The enzymatic activities of 3 ALAD mutants, K59N, A274T, and V153M, were 69.9%, 19.3%, and 41.0% of that of the wild-type ALAD, respectively, whereas 6 mutants, G133R, K59N/G133R, F12L, R240W, V275M, and delTC, showed little activity (< 8%). These variations generally reflect the phenotype of ALAD in vivo in patients with ADP and indicate that GST-ALAD fusion protein is indeed useful for predicting of the phenotype of ALAD mutants. The location of F12L mutation in the enzyme's molecular structure indicates that its disturbance of the quaternary contact of the ALAD dimer appears to have a significant influence on the enzymatic activity. Mouse monoclonal antibodies to human ALAD were developed that specifically recognized a carboxy terminal portion of ALAD, or other regions in the enzyme. This study represents the first complete analysis of 9 mutants of ALAD identified in ADP and indicates the highly heterogeneous nature of mutations in this disorder.

Molecular analysis of delta-aminolevulinate dehydratase deficiency in a patient with an unusual late-onset porphyria.

Cloning, expression, and genotype studies of the defective gene for delta-aminolevulinate dehydratase (ALAD) in a patient with an unusual late onset of ALAD deficiency porphyria (ADP) were carried out. This patient was unique in that he developed the inherited disease, together with polycythemia, at the age of 63. ALAD activity in erythrocytes of the patient was less than 1% of the normal control level. ALAD complementary DNA (cDNA) isolated from the patient's Epstein-Barr virus (EBV)-transformed lymphoblastoid cells had 2 base transitions in the same allele, G(177) to C and G(397) to A, resulting in amino acid substitutions K59N and G133R, respectively. It has been verified that the patient had no other ALAD mutations in this and in the other allele. By restriction fragment length polymorphism (RFLP) analysis, all family members of the proband who had one-half ALAD activity compared with the ALAD activity of the healthy control were shown to have the same set of base transitions. Expression of ALAD cDNA in CHO cells revealed that K59N cDNA produced a protein with normal ALAD activity, while G133R and K59N/G133R cDNA produced proteins with 8% and 16% ALAD activity, respectively, compared with that expressed by the wild type cDNA. These findings indicate that while the proband was heterozygous for ALAD deficiency, the G(397) to A transition resulting in the G133R substitution is responsible for ADP, and the clinical porphyria developed presumably due to an expansion of the polycythemic clone in erythrocytes that carried the mutant alad allele.

Regulation of beta-globin mRNA accumulation by heme in dimethyl sulfoxide (DMSO)-sensitive and DMSO-resistant murine erythroleukemia cells.

The level of mRNA encoding beta-globin was examined in dimethyl sulfoxide (DMSO)-sensitive (DS), and DMSO-resistant (DR) murine erythroleukemia (MEL) cells. DR cells lack erythroid-specific delta-aminolevulinate (ALA) synthase (AL-AS-E), and fail to undergo erythroid differentiation following treatment with DMSO. Treatment of cells with DMSO markedly increased ALAS-E mRNA in DS cells, while the same treatment downregulated the nonspecific ALA synthase (ALAS-N) mRNA levels in both DS and DR cells. The levels of beta-globin mRNA, heme content, and hemoglobin in DS cells increased, while those in DR cells decreased following treatment with DMSO. Treatment of DR cells with hemin caused an increase in beta-globin mRNA and hemoglobin, and partially restored the DMSO-mediated suppression of beta-globin mRNA and hemoglobin synthesis. DMSO treatment decreased heme oxygenase (HO) mRNA in hemin-treated DS cells, but not in hemin-treated DR cells. These findings indicate that heme is necessary for accumulation of the beta-globin transcript during erythroid differentiation, and that hemin-mediated HO induction becomes markedly downregulated in differentiated erythroid cells, presumably because less free heme is available for HO induction by a greater demand for the synthesis of hemoglobin.

Peptide and steroid hormones in subjects at different risk for diet-related diseases.

Populations eating low-fat or low-fat, high-fiber diets have lower mortality rates for many cancers and coronary heart disease. The importance of nutrient composition in the lumen on absorption and on function of the gastrointestinal tract as a factor in the development of these diseases has not been studied. We investigated the plasma levels of gut-CNS peptide hormones in lean and obese Dutch women fed a high-fat meal and administered cholecystokinin (CCK). After a high-fat meal the increase in plasma CCK was similar in lean and obese women. CCK administration significantly decreased insulin release in lean and obese women, decreased glucagon release in obese women, but caused a rapid increase in plasma glucagon in lean women. Although the CCK response was similar to a fat meal in lean and obese women, differences in the control of peptide hormone release occurred in response to fat meals and CCK administration.

Glucagon and insulin response to meals in non-obese and obese Dutch women.

Many digestive complaints are associated with abnormalities in gastrointestinal peptide hormone function. To investigate the effect of obesity on the release of pancreatic peptide hormones, we have compared the release of insulin and glucagon in non-obese-obese Dutch women in response to isocaloric mixed meals and to Naloxone, an opioid antagonist. Healthy premenopausal women who were separated into three groups based on body mass index (BMI less than 23; 23-27, greater than 28), were fed 600-calorie breakfasts. Higher fasting levels of plasma insulin and glucagon occurred in obese (BMI greater than 28) than lean (BMI less than 23) women, while glucagon and insulin release after a high fat meal occurred in obese women. Naloxone administration in obese women decreased plasma insulin and glucagon, but in lean women, naloxone increased plasma glucagon but did not alter plasma insulin levels. Results indicate differences in opiate effects on pancreatic function in non-obese-obese women.

Gut-CNS peptide hormones, digestive dysfunction, and colon cancer.

Dietary modification as protection against coronary heart disease and diet-related cancers has been recommended, yet the central nervous system (CNS)-neuroendocrine control of eating behavior and digestion is poorly understood. Maintenance of nutritional homeostasis (and therefore of an ideal body weight) requires a balance between appetite and satiety that currently appears to be related to CNS-peptide hormones stimulating feeding counteracted by the release of gut satiety peptide hormones. This review stresses the importance of the composition of luminal nutrients on colonic motility and the release of peptide hormones; it also discusses the interaction of the CNS and environmental factors on colonic activity and the relationship to diet-related diseases.

Plasma hormones in parous, nulliparous and postmenopausal Japanese women.

Mechanisms associated with protection against breast cancer in parous women with an early pregnancy are unknown. Elevated or deficient steroid hormone levels or dysfunctional release of prolactin have been postulated. Since these effects have not been reported in low-risk Japanese women, we have compared the hormone levels in age-weight-risk factor matched premenopausal nulliparous and parous Japanese women with established menstrual regularity. Higher luteal phase plasma levels of estradiol and estrone occurred in parous versus nulliparous women, while plasma prolactin and androgen levels were unaltered. It is suggested that the higher estrogen and lower LH levels, as in short versus long menstrual cycles, results from failure of the opioid GnRH. LH axis to return to pre-pregnancy levels.

VIP and prolactin release in response to meals.

To determine the role of vasoactive intestinal polypeptide (VIP) and prolactin (PRL) in digestion, plasma VIP and PRL levels were measured in healthy, non-obese and obese premenopausal women fed isocaloric breakfasts containing a high carbohydrate or a high fat-protein content. A significant increase in plasma VIP concentration occurred after a high fat-protein breakfast, the increase being independent of body mass index. Plasma PRL level was unaltered by breakfast. Results suggest that the release of VIP after a meal depends on a high fat concentration in the duodenum, which may then stimulate bicarbonate release and fat-stimulated release of cholecystokinin.

Gonadotrophin release and meat consumption in vegetarian women.

Many factors including diet modify the hypothalamic-pituitary axis and menstrual periodicity. We have determined the effect of a daily meat or a soybean supplement in rural vegetarian Black women on the length of the menstrual cycle and the episodic and luteinizing releasing hormone stimulated release of luteinizing hormone. The daily meat but not soybean supplement increased the length of the menstrual cycle (p less than or equal to 0.01), increased the release of LH (p less than or equal to 0.01), and decreased the stimulated release of LH in the luteal phase (p less than or equal to 0.01). These changes are opposite to those reported previously in the Caucasian women fed a meatless diet. Thus addition of meat in the diet modifies the episodic release of gonadotrophins and follicular maturation. The importance of a carbohydrate diet preferentially maintaining CNS-rhythmicity is suggested.

Quality control of lipid measurements in epidemiological studies: the U.S. Air Force HEART program.

A special standardization and quality assurance program, similar to that created for the Lipid Research Clinics Program (LRC), was developed for the American Health Foundation Laboratory by the Centers for Disease Control (CDC) to assure the quality of lipid measurements in the U.S. Air Force Health Evaluation and Risk Tabulation (HEART) Program. This study was designed to test the feasibility of reducing the incidence of heart disease in active-duty U.S. Air Force personnel through life-study intervention. During the 18-month study, CDC provided serum calibrators and reference materials for internal control and an external surveillance program for measurements of total cholesterol (TC) and high-density-lipoprotein cholesterol (HDLC). The Laboratory, using an automated enzymic method to measure cholesterol, achieved an overall goal for accuracy of less than 2% error (av systematic error, -30.6 mg/L) for TC, as measured on nine reference pools for which values were assigned by CDC. The average bias of measurements of HDLC with heparin-manganese to separate the lipoproteins in five CDC reference pools was -4.6 mg/L. Bias was estimated relative to the values assigned to the reference materials by the CDC reference methods for TC and HDLC. The average CV for TC was 0.89%, for HDLC 2.66%. Accuracy of cholesterol measurements can be assured over time with a standardization and quality-assurance program that incorporates accurately labeled reference materials for calibration, internal quality control, and external surveillance.

Diet and follicular development.

In Caucasian women, the length of the menstrual cycle is 28 days with a SD of 3.9 days. Many factors including diet modify menstrual periodicity. We have determined the effect of a meatless vegetarian diet in Caucasian women on the length of the menstrual cycle and the episodic and luteinizing releasing hormone-stimulated release of luteinizing hormone. The effect of a Western diet on the menstrual cycle in urban Black South African women was also studied. In Caucasian women, the vegetarian diet decreased (p less than or equal to 0.01) the pituitary response to luteinizing releasing hormone and decreased (p less than or equal to 0.05) the episodic release of luteinizing hormone. A Western diet increased (p less than or equal to 0.01) the duration of the follicular phase in Black South African women. Thus diet is a factor modifying the episodic release of gonadotrophins and follicular maturation.

Age, environmental factors and prostatic cancer.

Although prostatic cancer is evident late in life, pathological evidence suggests this disease is initiated earlier in life. As prostatic cancer is an endocrine associated disease and as adult hormone profiles are established during puberty, it was of interest whether difference in pubertal hormone levels occurred in populations at low or high risk for prostatic cancer. Accordingly we have investigated the hormone profiles in rural Black South African and urban white boys during puberty. It has been suggested that the timing of puberty is modified by environmental factors and that there is a concomitant control of gonadotrophin release and food intake by CNS-peptide hormones. It is therefore postulated that dietary factors during puberty modify the gut-CNS peptide hormones which in turn control the hypothalamic-pituitary-testicular axis. Distinct difference in plasma androgen and gonadotrophins between the two races are in part concordant with a modification of CNS-peptide hormones by environmental factors during puberty.

Effect of diet on plasma and urinary hormones in South African black men with prostatic cancer.

Epidemiological evidence suggests that the incidence and death rate from prostatic cancer, an endocrine-associated disease, are related to environmental factors including diet. In this study, a comparison of serum and urinary levels of steroid hormones was carried out in healthy elderly rural vegetarian South African black men, a low-risk population, and a comparable group of men with prostatic cancer. In these prostatic cancer patients, plasma androgen levels decreased, while estrogen levels increased. Concomitantly, the androsterone:etiocholanolone ratio increased, and a greater proportion of estrogens was excreted as estriol. When transferred to a Western diet, plasma androgens showed a further decrease and a greater increase in estrone in prostatic cancer patients. In prostatic cancer patients, the total urinary androgen and estrogen levels were unaltered. However, in elderly healthy men, the Western diet decreased the excretion of estrogens and androgens. Thus, a Western diet supplemented the decrease in plasma androgens initially present in these patients. Evidence suggests that the decrease in plasma androgens increases the estrogen: androgen ratio, which may lead to hyperplasia of the prostatic ductal epithelia, a change enhanced by a Western diet. Changes in urinary steroid hormone levels in South African black patients comparable to those reported in white prostatic cancer patients indicate that hormonal changes must be related to several environmental factors, apart from diet. A simultaneous study of the steroid hormone composition of blood and prostatic fluid in this low-risk population is suggested.

Response to luteinizing releasing hormone, thyrotrophic releasing hormone, and human chorionic gonadotropin administration in healthy men at different risks for prostatic cancer and in prostatic cancer patients.

A comparative study of the pituitary and testicular response to luteinizing releasing hormone (LHRH), thyrotrophic releasing hormone (TRH), and human chorionic gonadotrophin (HCG) administration was carried out in (a) low-risk young South African black men and high-risk North American black men for prostatic cancer and (b) healthy elderly South African men and South African black men with prostatic cancer. A comparable HCG response occurred in young South African and North American black men, while a greater release of prolactin, but a lesser release of luteinizing hormone in response to LHRH:TRH occurred in South African black men. The response to HCG was comparable in elderly and young South African black men, although the prolactin release in response to TRH was greater in elderly men. A more prolonged release of luteinizing hormone was evident in men with prostatic cancer. Higher estradiol and estrone but lower androstenedione levels occurred in men with prostatic cancer. Data suggest that, in the elderly South African black men with prostatic cancer, estrogen metabolism is modified and that either the estrogen level or the higher estrogen:androgen levels modify the pituitary response to LHRH:TRH. A Western diet enhanced the changes in hormone profiles evident in black South African men with prostatic cancer.

Environmental factors and breast and prostatic cancer.

Despite epidemiological evidence supporting a causal relationship between environmental factors and the development of breast and prostatic cancers, direct confirmation is unproven. Differences in hormone profiles in cancer patients and in patients with an increasing number of potential risk factors together with differences n life style and diet, suggest a relationship between diet, hormonal metabolism, and these endocrine-associated cancers. Modification of hormonal status by diet in men and women at different risk suggests that specific dietary components play a major role in these diseases.

Diet, lifestyle, and menstrual activity.

Menstrual activity is dependent on a critical body weight and may, therefore, be modified by nutritional factors. Lower plasma levels of testosterone, androstenedione, dehydroepiandrosterone, and prolactin, and differences in gonadotrophin levels were found during the menstrual cycle in rural black South African versus white women. When premenopausal vegetarian South African black women were fed a Western diet, plasma testosterone and prolactin levels increased, while estradiol decreased and follicle-stimulative hormone increased. In postmenopausal black women a similar diet decreased plasma leutinizing and follicle-stimulating hormone and increased prolactin levels. Differences between white and black women eating their customary diets may be related to genetic factors and/or lifestyle and diet. However, a Western diet induced changes in hormonal activity in vegetarian black women. These changes in hormonal levels in black women were comparable to those found in women with menstrual irregularities. Further study is needed to determine whether the difference in hormonal activity during the menstrual cycle between vegetarian black and white women may in part explain the lower incidence of coronary heart disease and breast cancer in the former.