Vitamin C: the known and the unknown and Goldilocks.

Vitamin C (Ascorbic Acid), the antiscorbutic vitamin, cannot be synthesized by humans and other primates, and has to be obtained from diet. Ascorbic acid is an electron donor and acts as a cofactor for fifteen mammalian enzymes. Two sodium-dependent transporters are specific for ascorbic acid, and its oxidation product dehydroascorbic acid is transported by glucose transporters. Ascorbic acid is differentially accumulated by most tissues and body fluids. Plasma and tissue vitamin C concentrations are dependent on amount consumed, bioavailability, renal excretion, and utilization. To be biologically meaningful or to be clinically relevant, in vitro and in vivo studies of vitamin C actions have to take into account physiologic concentrations of the vitamin. In this paper, we review vitamin C physiology; the many phenomena involving vitamin C where new knowledge has accrued or where understanding remains limited; raise questions about the vitamin that remain to be answered; and explore lines of investigations that are likely to be fruitful.

Phase I clinical trial of i.v. ascorbic acid in advanced malignancy.

BACKGROUND: Ascorbic acid is a widely used and controversial alternative cancer treatment. In millimolar concentrations, it is selectively cytotoxic to many cancer cell lines and has in vivo anticancer activity when administered alone or together with other agents. We carried out a dose-finding phase I and pharmacokinetic study of i.v. ascorbic acid in patients with advanced malignancies. PATIENTS AND METHODS: Patients with advanced cancer or hematologic malignancy were assigned to sequential cohorts infused with 0.4, 0.6, 0.9 and 1.5 g ascorbic acid/kg body weight three times weekly. RESULTS: Adverse events and toxicity were minimal at all dose levels. No patient had an objective anticancer response. CONCLUSIONS: High-dose i.v. ascorbic acid was well tolerated but failed to demonstrate anticancer activity when administered to patients with previously treated advanced malignancies. The promise of this approach may lie in combination with cytotoxic or other redox-active molecules.

A new recommended dietary allowance of vitamin C for healthy young women.

The recently released Recommended Dietary Allowance of vitamin C for women, 75 mg daily, was based on data for men. We now report results of a depletion-repletion study with healthy young women hospitalized for 186 +/- 28 days, using vitamin C doses of 30-2,500 mg daily. The relationship between dose and steady-state plasma concentration was sigmoidal. Only doses above 100 mg were beyond the linear portion of the curve. Plasma and circulating cells saturated at 400 mg daily, with urinary elimination of higher doses. Biomarkers of endogenous oxidant stress, plasma and urine F(2)-isoprostanes, and urine levels of a major metabolite of F(2)-isoprostanes were unchanged by vitamin C at all doses, suggesting this vitamin does not alter endogenous lipid peroxidation in healthy young women. By using Food and Nutrition Board guidelines, the data indicate that the Recommended Dietary Allowance for young women should be increased to 90 mg daily.

Reevaluation of ascorbate in cancer treatment: emerging evidence, open minds and serendipity.

Some clinicians and alternative therapy practitioners advocate megadose intravenous and oral ascorbate treatment of cancer. Randomized control studies using oral ascorbate showed no benefit. Recent data show that intravenous but not oral administration of ascorbate can produce millimolar plasma concentrations, which are toxic to many cancer cell lines. We propose that ascorbate treatment of cancer should be reexamined by rigorous scientific scrutiny in the light of new evidence.

Thyroid microcarcinoma: prevalence, prognosis, and management.

OBJECTIVE: To review the usual course of thyroid microcarcinoma (TMC) and the associated prognosis and treatment of affected patients. METHODS: We discuss predisposing factors in the formation of TMC and the modulation of its behavior, diagnostic evaluation, and management options. RESULTS: TMC, generally defined as a well-differentiated thyroid cancer less than or equal to 15 mm in diameter, has an estimated prevalence (based on autopsy studies) of about 5 to 10%. Studies, however, have shown that most of these cancers are smaller than 5 mm in diameter. The high prevalence of TMC in the general population contrasts with the rarity of thyroid cancers of greater size, which constitute less than 1% of malignant neoplasms in the United States. The frequent detection of TMC as a result of routine imaging of the neck for unrelated reasons and as a incidental finding in surgical specimens has raised a question about whether the management of TMC should differ from that for thyroid cancer of appreciable size. The uncertainty about optimal management of TMC is attributable to the small number of long-term follow-up studies as well as the common observation that patients usually have an excellent prognosis. Although in most patients harboring a TMC the cancer remains quiescent and never becomes clinically significant, in some cases TMC can demonstrate an aggressive course. Several variables, such as older age, multifocality, bilateral disease, and extrathyroidal spread at initial assessment, may have some adverse prognostic significance. After a partial surgical removal of the thyroid gland for TMC, the recurrence rate may be as high as 11%. Therefore, a treatment dilemma is caused by the low propensity of TMC for progression to clinically significant disease, yet the potential for recurrence and aggressive behavior in some cases. CONCLUSION: In general, surgical resection of TMC is based on results of fine-needle aspiration biopsy and the rate of growth of the nodule. Aggressive management seems indicated in high-risk patients, particularly older patients, those with a history of radiation exposure, and those with multifocal disease, bilateral disease, or lymph node involvement.

Cognitive dysfunction in patients with pituitary tumour who have been treated with transfrontal or transsphenoidal surgery or medication.

OBJECTIVE: This study was carried out to examine the neuropsychological status of patients treated for pituitary tumour by transfrontal surgery, transphenoidal surgery or medical treatment only, with or without radiotherapy. DESIGN AND MEASUREMENTS: Three groups of 23 patients who had been treated for pituitary tumour were compared with 23 healthy controls on a range of neuropsychological measures. The surgical patients were also subdivided into two groups and compared. The neuropsychological measures were standardized psychological tests designed to assess aspects of attention, memory and executive function. PATIENTS: The patients were those who had been treated with transfrontal surgery (n = 23), transsphenoidal surgery (n = 23) and medication only (n = 23). The groups did not differ with respect to age, education or premorbid ability level as assessed by the National Adult Reading Test. All participants were free of known sources of cognitive impairment other than pituitary tumour. RESULTS: Comparison of the four groups revealed that nearly half of the transfrontal, one-third of the transsphenoidal and one-quarter of the non-surgical group had three or more neuropsychological tests scores below the 10th percentile compared to less than 5% of the controls. Impairments in memory and executive function were found in both surgical groups. The non-surgical patients appeared to have problems only on tasks requiring high levels of cognitive processing. Differences were found between the two surgical groups with respect to the severity of the cognitive impairment, the transfrontal patients having more severe impairment than the transsphenoidal. No significant negative effects on cognitive functioning were associated with radiotherapy; however, transfrontal surgery patients who had not been treated with radiotherapy were found to be more impaired than other patients. This was thought to be related to radical surgery. CONCLUSIONS: Many patients with treated pituitary tumour suffer significant cognitive impairment. The severity and nature of impairment differs between treatment groups, although the cause of this could not be addressed by this study. Recommendations are made for future research and clinical practice.

Bilateral sequential inferior petrosal sinus sampling with corticotrophin-releasing hormone stimulation in the diagnosis of Cushing's disease.

OBJECTIVE: The demonstration of a central to peripheral ACTH gradient in a hypercortisolaemic patient is diagnostic of Cushing's disease. We tried to determine whether single blood samples for ACTH obtained sequentially from each of the inferior petrosal sinuses following human corticotrophin-releasing hormone (hCRH) stimulation can reliably establish such a gradient. DESIGN: Prospective study. PATIENTS: Seventeen patients with clinical and biochemical features of Cushing's syndrome. METHODS: After the administration of hCRH, the patients underwent bilateral sequential inferior petrosal sinus sampling, with a single blood sample obtained from each of the inferior petrosal sinuses sequentially, along with a peripheral venous sample. The petrosal sinus catheter was withdrawn immediately after obtaining a blood sample. Patients did not require indwelling catheters in the petrosal sinuses, nor heparinisation. RESULTS: Bilateral sequential inferior petrosal sinus sampling correctly identified a pituitary source of ACTH, as shown by a central to peripheral ACTH ratio >2, in all patients in whom the procedure was successfully carried out. All patients underwent transsphenoidal pituitary surgery resulting in remission. CONCLUSIONS: The simplified method of inferior petrosal sinus sampling, using a single sequential sample from each of the inferior petrosal sinuses, following initial hCRH stimulation, is as accurate as the more complex test using multiple bilateral simultaneous inferior petrosal sinus samples. It avoids the use of indwelling cerebral venous catheters and is therefore unlikely to cause brain stem damage.

Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP.

The goals of this work were to establish a reproducible and effective model of apoptosis in a cell line derived from advanced prostate cancer and to study the role of the caspase family of proteases in mediating apoptosis in this system. The study involved the use of the prostate cancer cell line LNCaP. Apoptosis was induced using the hydroxymethyl glutaryl CoA reductase inhibitor, lovastatin, and was evaluated by agarose gel electrophoresis of genomic DNA, morphological criteria, and terminal deoxynucleotidyl transferase-mediated nick end labeling. Caspases were studied by catalytic activity, mRNA induction, and protein processing. Lovastatin (30 microM) was an effective inducer of apoptosis, causing changes that were evident after 48 h and essentially complete after 96-120 h of treatment. These effects were prevented by the simultaneous addition of mevalonate (300 microM) to the culture medium. Lovastatin induced a proteolytic activity that was able to cleave the enzyme poly(ADP-ribose) polymerase and the substrate Z-DEVD-AFC, which is modeled after the P1-P4 amino acids of the poly(ADP-ribose) polymerase cleavage site. Caspase-7, but not caspase-3, underwent proteolytic activation during lovastatin-induced apoptosis, an effect prevented by mevalonate. Caspase-7 was the only detected interleukin 1beta converting enzyme family protease with DEVD cleavage activity that exhibited lovastatin-induced mRNA up-regulation. Again, mevalonate blocked this effect. Lovastatin-induced apoptosis also was prevented when the caspase inhibitors Z-DEVD-CH2F or Z-VAD-CH2F (100 microM) where added to the medium. These studies have identified lovastatin as a powerful inducer of apoptosis in the cell line LNCaP. Caspase activation was a necessary event for LNCaP cells to undergo apoptosis during treatment with lovastatin. Of the caspases tested, only caspase-7 underwent proteolytic activation after stimulation with lovastatin. Identification of caspase-7 as a potential mediator of lovastatin-induced apoptosis broadens our knowledge of the molecular events associated with programmed cell death in a cell line derived from prostatic epithelium.

Erythropoietin levels can be used to determine tissue oxygenation in critically ill patients.

Critically ill patients often require pharmacological support to maintain blood pressure and cardiac output, and mechanical ventilation to ensure adequate oxygenation. Current methods of measurement of oxygen delivery do not necessarily reflect tissue oxygenation. Levels of circulating erythropoietin reliably reflect the adequacy of renal oxygen supply under physiological and many pathological conditions. It is proposed that the measurement of circulating erythropoietin can be used as a measure of tissue oxygenation in critically ill patients.

Lovastatin-induced apoptosis in prostate stromal cells.

Benign prostatic hyperplasia (BPH) is a common disease of aging men. Current medical treatment for this condition is only partially effective, therefore many patients must undergo surgery for symptomatic relief. BPH is caused by an increase in prostate epithelial and stromal cells, especially the latter. Since BPH stromal cells have a long life span and are not very responsive to androgen withdrawal, cultured BPH stromal cells were used to explore the feasibility of pharmacologically inducing apoptosis in these cells. We obtained BPH tissue during surgery, and stromal cells were isolated and maintained in culture. After cells achieved confluence, we induced apoptosis with the HMGCoA reductase inhibitor, lovastatin (30 micromol/L). The effects of testosterone (100 micromol/L), dihydrotestosterone (DHT; 100 micromol/L) and finasteride (100 micromol/L) on lovastatin-induced apoptosis were studied on cells grown in media containing charcoal stripped serum. Similarly, we examined the effect of the cholesterol pathway metabolites, mevalonic acid (30 micromol/L), geranyl geraniol (30 micromol/L), farnesol (10 micromol/L), squalene (30 micromol/L) and 7-ketocholesterol (3 micromol/L) on lovastatin-induced apoptosis. We demonstrated apoptosis by DNA laddering in agarose gels, by fluorescence microscopy following acridine orange staining, and by flow cytometry after end-labeling of DNA strand breaks with biotin-16-dUTP using deoxynucleotidyl exotransferase (TdT). Lovastatin at 30 micromol/L, but not at lower concentrations, induced apoptosis in BPH prostate stromal cells. This was seen (by flow cytometry) in 16.6 +/- 7.3% (mean +/- SD) of BPH cells treated with lovastatin at 72 h vs. 2.5 +/- 1.2% of cells treated with ethanol. Lovastatin-induced apoptosis was not increased in stripped serum or by the addition finasteride, and was not inhibited by testosterone or DHT. Only mevalonate and geranyl geraniol, prevented lovastatin-induced apoptosis whereas farnesol, squalene, or 7-ketocholesterol did not. We conclude that lovastatin can induce apoptosis in BPH stromal cells in vitro, and this is not affected by androgen withdrawal or stimulation. It is unlikely that lovastatin, per se, will be an effective treatment for BPH in vivo, but it does provide a means for inducing apoptosis in vitro. Understanding the apoptotic process in BPH stromal cells ultimately may lead to new therapeutic strategies for BPH.

Octreotide treatment increases exercise capacity in patients with acromegaly.

This prospective study was conducted to determine the effect of Octreotide treatment on cardiovascular function in patients with active acromegaly. Ten acromegalic patients who failed to suppress growth hormone (GH) to < 5 mU/l during a 2 h oral glucose tolerance test were treated with 100 micrograms of Octreotide subcutaneously three times daily for 2 months, followed by 200 micrograms three times daily if the mean GH level was > 5 mU/l, for a total of 1 year. All patients had GH and insulin-like growth factor I (IGF-I) estimation, ejection fraction determined by Echocardiogram and multigated image acquisition scan, electrocardiogram (ECG), exercise ECG, 24-h ECG and chest x-ray. At 6 and 12 months, both GH and IGF-I were reduced but ECG, heart size and ejection fraction were unchanged. The patients improved symptomatically and had significant reduction in resting heart rate and increase in weight. Exercise time (mean +/- SD) increased from 637 +/- 137s at baseline to 787 +/- 101s at 1 year (p < 0.01) and work done increased from 9 +/- 3.3 to 11.9 +/- 2.7 metabolic equivalents (p < 0.001). We conclude that the decrease in GH and IGF-I following Octreotide treatment of acromegaly is accompanied by decreased heart rate and increased exercise capacity despite an unchanged ejection fraction.

The effects of insulin-like growth factor-1 on plasminogen activator inhibitor-1 synthesis and secretion: results from in vitro and in vivo studies.

In vitro studies have shown that insulin and IGF-1 releases the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1) from cells of hepatic origin. To investigate the effects of IGF-1 on fibrinolysis: 1) cultured hepatoma cells were grown in the presence of IGF-1 and media collected for secreted PAI-1 and cells probed for PAI-1 mRNA, 2) 8 hypopituitary patients were treated with recombinant human growth hormone (rhGH) and 3) 5 type 2 diabetic patients were treated with recombinant human IGF-1 (rhIGF-1). Treatment of Hep G2 cells with IGF-1 (1000 ng/ml) increased secretion of PAI-1 from a median value of 80 ng/10(6) cells (range 21-91) to 144 ng/10(6) cells (range 128-169) after 24 h (p < 0.01). Synthesis of PAI-1 mRNA increased in a similar fashion. Treatment of hypopituitary patients with rhGH led to an increase in circulating IGF-1 from a mean value of 166 (range 41-324) ng/ml at baseline to 322 (77-575) ng/ml at 4 weeks and 259 (104-533) ng/ml after 8 weeks (p < 0.02). Despite this, no changes in circulating PAI-1 or fibrinolysis occurred. Type II diabetic patients treated with rhIGF-1 showed an increase in circulating IGF-1 from a mean value of 120 ng/ml (range 109-196), at baseline to 823 ng/ml (585-894) after 5 days. This also was not associated with changes in circulating PAI-1 or in fibrinolysis. The results confirm that IGF-1 induces the synthesis of PAI-1 in Hep G2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)