Attallah Kappas

Pharmacological and Clinical Aspects of Heme Oxygenase

This review is intended to stimulate interest in the effect of increased expression of heme oxygenase-1 (HO-1) protein and increased levels of HO activity on normal and pathological states. The HO system includes the heme catabolic pathway, comprising HO and biliverdin reductase, and the products of heme degradation, carbon monoxide (CO), iron, and biliverdin/bilirubin. The role of the HO system in diabetes, inflammation, heart disease, hypertension, neurological disorders, transplantation, endotoxemia and other pathologies is a burgeoning area of research. This review focuses on the clinical potential of increased levels of HO-1 protein and HO activity to ameliorate tissue injury. The use of pharmacological and genetic probes to manipulate HO, leading to new insights into the complex relationship of the HO system with biological and pathological phenomena under investigation, is reviewed. This information is critical in both drug development and the implementation of clinical approaches to moderate and to alleviate the numerous chronic disorders in humans affected by perturbations in the HO system.

Studies in lead poisoning: II. Correlation between the ratio of activated to inactivated δ-aminolevulinic acid dehydratase of whole blood and the blood lead level☆

Abstract 1. 1. A method is described, using aliquots of 5 μl heparinized blood, to determine the activity of δ-aminolevulinic acid dehydratase in the aliquot incubated with δ-aminolevulinic acid, and in the aliquot incubated with δ-aminolevulinic acid + dithiothreitol. 2. 2. The log of the activity of δ-aminolevulinic acid dehydratase in the absence of dithiothreitol is inversely correlated with the blood lead level with a correlation coefficient of 0.72. 3. 3. The ratio: dithiothreitol-activated enzyme/nonactivated enzyme, vs the blood lead level gives a linear regression line with the higher correlation coefficient of 0.8. 4. 4. Dithiothreitol (20 m M ) fully activates the enzyme in lead-poisoned blood to the levels of activated normal blood. Thus lead 0.09 mg 100 ml blood does not appear to inhibit the formation, or affect the destruction of δ-aminolevulinic acid dehydratase. 5. 5. Kinetic analysis indicates that lead changes the affinity of the dehydratase for δ-aminolevulinic acid (i.e., K m ) slightly, but markedly lowers the V max , i.e., the inhibition is primarily of the noncompetitive type.

Human Heme Oxygenase-1 Gene Transfer Lowers Blood Pressure and Promotes Growth in Spontaneously Hypertensive Rats

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, with release of free iron and carbon monoxide. Both heme and carbon monoxide have been implicated in the regulation of vascular tone. A retroviral vector containing human HO-1 cDNA (LSN-HHO-1) was constructed and subjected to purification and concentration of the viral particles to achieve 5×109 to 1×1010 colony-forming units per milliliter. The ability of concentrated infectious viral particles to express human HO-1 (HHO-1) in vivo was tested. A single intracardiac injection of the concentrated infectious viral particles (expressing HHO-1) to 5-day-old spontaneously hypertensive rats resulted in functional expression of the HHO-1 gene and attenuation of the development of hypertension. Rats expressing HHO-1 showed a significant decrease in urinary excretion of a vasoconstrictor arachidonic acid metabolite and a reduction in myogenic responses to increased intraluminal pressure in isolated arterioles. Unexpectedly, HHO-1 chimeric rats showed a simultaneous significant proportionate increase in somatic growth. Thus, delivery of HHO-1 gene by retroviral vector attenuates the development of hypertension and promotes body growth in spontaneously hypertensive rats.

Studies in lead poisoning: I. Microanalysis of erythrocyte protoporphyrin levels by spectrofluorometry in the detection of chronic lead intoxication in the subclinical range☆

Abstract This study describes the application of a rapid, simple, extremely sensitive new assay for blood protoporphyrin to the detection of lead poisoning in the subclinical range. This protoporphyrin method has proved to be especially useful for the detection of chronic lead intoxication in children because of its precision and the fact that only 2 μl of blood are required for the analysis. Blood protoporphyrin was found to reflect better the level of bone marrow lead existing 2–3 months prior to sampling rather than the circulating blood lead level. The correlation coefficient between log blood protoporphyrin and blood lead was found to be 0.72. Over 95% of the population with a blood lead level of 0.06 mg/100 ml had a protoporphyrin concentration of ≧ 140 μg/100 ml RBC. When a group of children was selected whose blood lead was in equilibrium with bone marrow lead, the correlation coefficient was 0.91. Several micromethods for the detection of lead poisoning are discussed; using a combination of this new protoporphyrin assay together with either the determination of blood lead, the presence of increased eythrocytic osmotic resistance or the measurement of δ-aminolevulinic acid dehydratase, permits a distinction to be made between acute and chronic lead poisoning and/or iron deficiency anemia.

Drug metabolism by the human hepatoma cell, Hep G2

The human liver-derived cell line, Hep G2, has aryl hydrocarbon hydroxylase and 7-ethoxycoumarin o-de-ethylase activities. Partial purification of cytochrome P-450 from Hep G2 cells provided spectral evidence of this hemeprotein in the purified fraction. These results suggest that Hep G2 cells will be useful for the study of cytochrome P-450 and the regulation of mixed function oxidase activities in liver cells of human origin.

Diet-hormone interactions: Protein/carbohydrate ratio alters reciprocally the plasma levels of testosterone and cortisol and their respective binding globulins in man

The aim of this study was to determine if a change in protein/carbohydrate ratio influences plasma steroid hormone concentrations. There is little information about the effects of specific dietary components on steroid hormone metabolism in humans. Testosterone concentrations in seven normal men were consistently higher after ten days on a high carbohydrate diet (468 +/- 34 ng/dl, mean +/- S.E.) than during a high protein diet (371 +/- 23 ng/dl, p less than 0.05) and were accompanied by parallel changes in sex hormone binding globulin (32.5 +/- 2.8 nmol/l vs. 23.4 +/- 1.6 nmol/l respectively, p less than 0.01). By contrast, cortisol concentrations were consistently lower during the high carbohydrate diet than during the high protein diet (7.74 +/- 0.71 micrograms/dl vs. 10.6 +/- 0.4 micrograms/dl respectively, p less than 0.05), and there were parallel changes in corticosteroid binding globulin concentrations (635 +/- 60 nmol/l vs. 754 +/- 31 nmol/l respectively, p less than 0.05). The diets were equal in total calories and fat. These consistent and reciprocal changes suggest that the ratio of protein to carbohydrate in the human diet is an important regulatory factor for steroid hormone plasma levels and for liver-derived hormone binding proteins.

Enhanced phenacetin metabolism in human subjects fed charcoal‐broiled beef

There were marked individual differences in the plasma levels of phenacetin after oral administration of a 900‐mg dose to 9 normal volunteers eating their customary home diet. Feeding a diet that contained charcoal‐broiled beeffor 4 days prior to the administration of phenacetin markedly decreased the plasma levels of this drug without appreciably influencing the plasma concentrations of phenacetins metabolite, N‐acetyl‐p‐aminophenol (APAP), or the plasma half‐life of phenacetin. The average peak concentration of phenacetin in plasma, after a 900‐mg oral dose, fell from 1,628 ng/ml, when the subjects were fed a control diet for 7 days, to 352 ng/ml after they were fed the same diet which contained charcoal‐broiled beeffor 4 days. The average peak concentration of phenacetin rose to 1,885 ng Iml after the subjects were subsequently fed the control diet for 7 days. The ratios of the average concentrations of APAP in plasma to those of phenacetin markedly increased after the charcoal‐broiled beef diet. The results suggest that a diet containing charcoal‐broiled beef enhances the metabolism of phenacetin in the gastrointestinal tract and/or during its first pass through the liver. This effect greatly decreases the bioavailability of phenacetin.

Heme Oxygenase-1 Induction Remodels Adipose Tissue and Improves Insulin Sensitivity in Obesity-Induced Diabetic Rats

Obesity-associated inflammation causes insulin resistance. Obese adipose tissue displays hypertrophied adipocytes and increased expression of the cannabinoid-1 receptor. Cobalt protoporphyrin (CoPP) increases heme oxygenase-1 (HO-1) activity, increasing adiponectin and reducing inflammatory cytokines. We hypothesize that CoPP administration to Zucker diabetic fat (ZDF) rats would improve insulin sensitivity and remodel adipose tissue. Twelve-week-old Zucker lean and ZDF rats were divided into 4 groups: Zucker lean, Zucker lean–CoPP, ZDF, and ZDF–CoPP. Control groups received vehicle and treatment groups received CoPP (2 mg/kg body weight) once weekly for 6 weeks. Serum insulin levels and glucose response to insulin injection were measured. At 18 weeks of age, rats were euthanized, and aorta, kidney, and subcutaneous and visceral adipose tissues were harvested. HO-1 expression was measured by Western blot analysis and HO-1 activity by serum carbon monoxide content. Adipocyte size and cannabinoid-1 expression were measured. Adipose tissue volumes were determined using MRI. CoPP significantly increased HO-1 activity, phosphorylated AKT and phosphorylated AMP kinase, and serum adiponectin in ZDF rats. HO-1 induction improved hyperinsulinemia and insulin sensitivity in ZDF rats. Subcutaneous and visceral adipose tissue volumes were significantly decreased in ZDF rats. Adipocyte size and cannabinoid-1 expression were both significantly reduced in ZDF–CoPP rats in subcutaneous and visceral adipose tissues. This study demonstrates that HO-1 induction improves insulin sensitivity, downregulates the peripheral endocannabinoid system, reduces adipose tissue volume, and causes adipose tissue remodeling in a model of obesity-induced insulin resistance. These findings suggest HO-1 as a potential therapeutic target for obesity and its associated health risks.

Effect of charcoal‐broiled beef on antipyrine and theophylline metabolism

Eight healthy volunteers were sequentially fed a control diet, a charcoal‐broiled beef‐containing diet, and the control diet a second time. The mean plasma half‐lives (t½) of antipyrine and theophylline were each decreased by 22% after the subjects were fed the charcoal‐broiled beef‐containing diet. The mean plasma t½s for these drugs returned to control values when the subjects were fed the control diet for a second time. Considerable individuality occurred in the responsiveness of the subjects to the charcoal‐broiled beef‐containing diet. The decreases in antipyrine plasma t½s among the 8 subjects ranged from 5% to 39%, and the decreases in theophylline t½S ranged from 0% to 42%.

Hereditary Tyrosinemia and the Heme Biosynthetic Pathway. PROFOUND INHIBITION OF δ-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY BY SUCCINYLACETONE

Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of delta-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 x 10(-7) M to 3 x 10(-7) M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic consequences that mimic another genetic disease, AIP.