Diagnosis of essential hypertension in humans by the determination of plasma renal cortexin using enzyme-linked immunosorbent assay.

BACKGROUND: More than 90% of all hypertensive persons are reported to have essential hypertension (EH), a particular form of elevated blood pressure, for which no diagnostic test is currently available. The level of plasma renal (R) cortexin (PRC), a hypotensive protein produced in the kidney cortex cells, was reported to be reduced from 218 nM in the plasma of normotensive persons (NP) to 0 nM in the plasma of patients with EH. The feasibility of using the determination of PRC by enzyme-linked immunosorbent assay (ELISA) as a diagnostic test for EH was investigated. METHODS: The PRC was determined by ELISA using electrophoretically pure cortexin as the antigen. A total of 344 persons (male and female) with EH, with or without diabetes mellitus (DM), and receiving or not receiving any anti-hypertensive and/or anti-diabetic medication at presentation, as well as an equal number of age- and gender-matched NP participated in the study. RESULTS: All persons with EH, with or without co-existing DM, were found to have 0 nM PRC, regardless of whether they were receiving anti-hypertensive and anti-diabetic drugs, including those who had been taking these medications over an extended period of time (3 months). CONCLUSIONS: The determination of PRC as a marker protein by ELISA, a rapid method that can be carried out in any diagnostic laboratory, was shown to be suitable for the diagnosis of EH, even in those subjects who had co-existing DM and were receiving both anti-hypertensive and anti-diabetic medication.

The appearance of dermcidin isoform 2, a novel platelet aggregating agent in the circulation in acute myocardial infarction that inhibits insulin synthesis and the restoration by acetyl salicylic acid of its effects.

Hyperglycemia with severe reduction of plasma insulin level is frequently associated with acute ischemic heart disease. Since insulin is reported to be an anti thrombotic humoral factor, the mechanism of the impaired insulin synthesis was investigated. The plasma from the patients with acute myocardial infarction (AMI) was analyzed by SDS-polyacrylamide gel electrophoresis. Dermcidin isoform 2 (dermcidin) was determined by enzyme linked immunosorbent assay. Insulin synthesis was determined by in vitro translation of glucose induced insulin mRNA synthesis in the pancreatic ß cells. Nitric oxide (NO) was determined by methemoglobin method. SDS-polyacrylamide gel electrophoresis of AMI plasma demonstrated the presence of a novel protein band of Mr 11 kDa that was determined to be dermcidin. Addition of 0.1 µM dermcidin inhibited insulin synthesis by >65 fold compared to control through the inhibition of NO synthesis in the pancreatic cells. The oral administration of 150 mg acetyl salicylic acid (aspirin) to the AMI patients increased the plasma insulin level from 13 (median) to 143 µunits/dl (median) with concomitant decrease of plasma dermcidin level from 112 to 9 nM in these patients within 12 h. It was also found that while the injection of 3.0 ± 0.05 (n = 10) nmol dermcidin with 0.25 ± 0.03 µmol ADP/g body weight caused coronary thrombus in mice, ADP itself at this concentration failed to produce thrombus. These results indicated that dermcidin was a novel platelet aggregating agent, and potentiated the ADP induced thrombosis in the animal model as well as acutely inhibited glucose induced insulin synthesis.

Acetyl salicylic acid (aspirin) improves synthesis of maspin and lowers incidence of metastasis in breast cancer patients [corrected].

Maspin, a 42 kDa protein produced in normal breast cells, has been shown to inhibit the invasion and metastasis of breast cancer in an animal model. Ingestion of acetylsalicylic acid (aspirin) by breast cancer patients has been reported to restore the systemic synthesis of maspin through the stimulation of systemic nitric oxide production. Studies were carried out to determine the effect of aspirin on the incidence of breast cancer metastasis, which is reported to occur in 50% of patients who have previously received chemotherapy, radiation, and/or surgery. Thirty-five female patients (aged 41-65 years) with breast cancer who had previously received these therapies took one 75 mg/70 kg body weight enteric-coated aspirin tablet every 24 h, after an adequate meal, for 3 years. Their plasma nitric oxide and maspin levels were measured. The occurrence of metastasis was ascertained monthly by a qualified oncologist, and confirmed, if necessary, by biopsy. Daily ingestion of aspirin by participants resulted in an increase in maspin levels from 0.95 ± 0.04 to 4.63 ± 0.05 nM after 24 h. These levels were maintained for 3 years. These studies suggest that daily ingestion of aspirin might significantly reduce the incidence of breast cancer metastasis in patients who have previously received anticancer therapies.

The role of leucocytes in the acetyl salicylic acid (aspirin) induced nitric oxide synthesis in the production of interferon-alpha, a potent inhibitor of platelet aggregation and a thrombolytic agent.

The role of aspirin-induced NO synthesis in the production of interferon-alpha (IFN-alpha) in leucocytes and the effect of IFN-alpha on platelet aggregation was studied. Treatment of Platelet Rich Plasma (PRP) with the dialyzed supernatant from the leucocyte suspension incubated with 80 microM aspirin resulted in parallel syntheses of NO and IFN-alpha as determined by methemoglobin assay and enzyme linked immunosorbent assay respectively. Incubation of PRP with 10 nM purified IFN-alpha for 40 min resulted in the maximal inhibition of platelet aggregation through the synthesis of NO due to the activation of nitric oxide synthase in platelets by IFN-alpha. The treatment of clotted PRP with IFN-alpha resulted in the lysis of the clot due to the fibrinolysis. Injection of IFN-alpha was found to protect mice from death due to the lysis of ADP-induced coronary thrombus. Interferon-alpha was found to be a potent inhibitor of platelet aggregation and a thromboprotective agent.

Systemic production of IFN-alpha by garlic (Allium sativum) in humans.

The effect of foods on the production of interferon-alpha (IFN-alpha) is currently unknown. Garlic (Allium sativum) used as a folk medicine is reported to stimulate nitric oxide (NO) production. We investigated the systemic increase of NO due to the ingestion of garlic on the plasma IFN-alpha level in normal volunteers. Normal volunteers (10 groups, 10 in each group) ate 2 g fresh garlic, and plasma NO and IFN-alpha levels were determined after 2 and 4 h. The participants were also asked to eat garlic for various periods of time, and plasma NO and IFN-alpha were similarly assayed. Ingestion of 2 g fresh, but not boiled, garlic was found to increase the basal plasma level of NO from 2.7 +/- 0.1 microM to 8.76 +/- 0.21 microM at 2 and 4 h, respectively. The basal plasma IFN-alpha level increased from 9.51 +/- 0.26 nM to 46.3 +/- 1.2 nM in normal volunteers (n = 10) at the same time. The chronic eating of garlic was found to maintain IFN-alpha at high levels for at least 7 days. The exposure of neutrophils to garlic in vivo or in vitro, which also stimulated synthesis of NO in these cells, was found to stimulate IFN-alpha synthesis as measured by the stimulation of IFN-alpha mRNA synthesis. Thus, consumption of garlic resulted in stimulated synthesis of NO and, in turn, IFN-alpha in humans, which could be beneficial in viral or proliferative diseases.

Effect of progesterone receptor status on maspin synthesis via nitric oxide production in neutrophils in human breast cancer.

BACKGROUND: Although progesterone receptor (PR) status, similarly to estrogen receptor status, is of prognostic importance in breast cancer, the involvement of the PR in breast cancer remains obscure. Studies were conducted to determine the function of the PR in neutrophils in the nitric oxide-induced synthesis of maspin, an anti-breast-cancer protein produced in nonmalignant mammary cells and in neutrophils in the circulation. METHODS: PR status was determined by immunohistochemistry. Maspin synthesis was determined by in-vitro translation of messenger RNA and quantified by enzyme-linked immunosorbent assay. Nitric oxide was determined by the methemoglobin method. RESULTS: It was found that PR status in neutrophils was identical with that in malignant breast tissues. A Scatchard plot for progesterone binding to normal and PR-positive (PR+) neutrophils revealed that whereas normal neutrophils had 11.5 × 10(10) PR sites/cell with K d = 47.619 nM, PR+ neutrophils had 6.6 × 10(10) PR sites/cell with K d = 47.619 nM. The progesterone negative (PR-) neutrophils failed to bind to progesterone. Incubation of normal and PR+ neutrophils with 25 nM progesterone produced 1.317 µM NO and 2.329 nM maspin; the PR+ neutrophils produced 0.72 µM NO and 1.138 nM maspin. The PR- neutrophils failed to produce any NO or maspin in the presence of progesterone. Inhibition of progesterone-induced NO synthesis led to complete inhibition of maspin synthesis in all neutrophils. CONCLUSION: These results suggest that estrogen and progesterone complement each other in NO-induced maspin synthesis, and do not necessarily antagonize in the synthesis of the anti-breast-cancer protein.

The "Cross Talk" between the Receptors of Insulin, Estrogen and Progesterone in Neutrophils in the Synthesis of Maspin through Nitric Oxide in Breast Cancer.

PURPOSE: The binding of either insulin, or estrogen or progesterone to their specific receptors on neutrophils has been reported to stimulate nitric oxide (NO) induced maspin synthesis in these cells. Experiments were carried out to determine the role of insulin receptor interaction in the nitric oxide induced maspin synthesis in neutrophils that was effected by estrogen or progesterone. METHODS: Estrogen receptor positive (ER+) and progesterone receptor positive (PR+) neutrophils were isolated from the blood cancer subjects. Maspin was determined by enzyme linked immunosorbent assay after in vitro translation of maspin mRNA. NO was determined by methemoglobin method. RESULTS: It was found that pre incubation of normal neutrophils with insulin to reach equilibrium binding decreased both ER and PR numbers by ≈50% without changing the dissociation constants of the steroids binding. The reduction of ER or PR numbers on neutrophils due to the pretreatment with insulin resulted in the decreased NO induced maspin synthesis from 2.383 ± 0.014 nM to 1.454 ± 0.004 nM in the case of estrogen and in the decrease of maspin synthesis from 2.329 ± 0.012 nM to 1.410 ± 0.002 nM in the case of progesterone. The incubation of ER+ neutrophils or PR+ neutrophils with insulin further decreased the maspin synthesis from 1.422 ± 0.029 nM to 0.790 ± 0.004 nM in the case of estrogen, and from 1.138 ± 0.024 nM to 0.555 ± 0.003 nM maspin in the case of progesterone respectively compared to normal control. CONCLUSION: These results suggested that a "cross-talk" between the insulin receptors and the steroid receptors down regulated maspin synthesis in normal and in breast cancer neutrophils.

The Role of Neutrophil Estrogen Receptor Status on Maspin Synthesis via Nitric Oxide Production in Human Breast Cancer.

PURPOSE: Estrogen, through its binding to nuclear estrogen receptor (ER), has been implicated in the development of human breast cancer. The presence or absence of ER in breast lesions has been used to classify breast cancer into ER+ or ER- type. Maspin, an anti-breast cancer protein produced in normal mammary cells, has also been reported to control the condition. Studies have been conducted to determine the role of ER+ and ER- status in neutrophils in the synthesis of maspin in human breast cancer. METHODS: Maspin presence was determined by enzyme linked immunosorbent assay, while nitric oxide (NO) level was determined using the methemoglobin method. RESULTS: Scatchard plots of the equilibrium binding of estrogen demonstrated the presence of 4.18×10(7) receptors per normal neutrophil and 2.46×10(7) receptors per ER+ neutrophil with a similar dissociation constant (0.926 nM). The ER- type showed nonspecific estrogen binding only. At 0.6 nM estrogen, NO synthesis was maximally increased to 1.829 and 0.887 µM NO/10(9) cells at 4 hours in normal and ER+ neutrophils respectively, with synthesis of 2.383 and 1.422 nM maspin in normal and ER+ neutrophils respectively. Estrogen failed to produce these effects in ER- neutrophils. CONCLUSION: ER status in neutrophils determined maspin synthesis in breast cancer through the stimulation of NO synthesis. Neutrophils with ER- status which do not produce any maspin when treated with estrogen, might imply a worse prognostic outcome in ER- breast cancer due to the lack of anti-breast cancer protein synthesis.