|LE Magazine September 1999 |
Postsurgical therapy and wound healing
In order to analyze the possible role of pantothenic acid (PA) and ascorbic acid (AA) in wound healing processes, the effects of these vitamins upon the growth of fibroblasts, obtained from human fetal skin or foreskin, were studied. Cell proliferation, protein synthesis and protein release were evaluated. The rate of cell growth remained identical when PA or AA were added to the culture medium. PA increased the basal incorporation of 14C proline into precipitated material while AA did not modify this action. However, when cultures were incubated with PA and AA, the release of intracellular protein into the culture medium increased. These results suggest that the combined use of these two vitamins might be of interest in postsurgical therapy and in wound healing.
Antioxidants and ischemic skin wounds
Oxygen free radicals are produced and play an important role in ischemic injury. We therefore wished to investigate the role of free radicals on ischemic skin wound healing. For this purpose, H-shaped flaps, where the test ischemic wound is the horizontal line in the H, were created on the dorsum of the rat. To inhibit the probable hazards of free radicals, allopurinol and superoxide dismutase (SOD) were given to the animals. Most of the studied wound-healing parameters were impaired in the ischemic group. In the allopurinol-treated group, breaking strength was increased by 52% by day 7 and by 109% by day 14 (p < 0.0002 and p < 0.001), and in the SOD-treated group the increase was 69% both by days 7 and 14 of healing when compared with the ischemic control group (p < 0.003 and p < 0.002). Hydroxyproline content was increased 75% with allopurinol and 113% with SOD in the wound by day 7 (p < 0.03 and p < 0.001 respectively). SOD treatment caused a significant decrease in wound edema by day 7 of healing (p < 0.05). Histopathological evaluation revealed that in the SOD- and allopurinol-treated groups, the amount of collagen and its organization were more prominent when compared with the ischemic controls. These results show that oxygen free radicals play an important role in the failure of ischemic wound healing, and antioxidants partly improve the healing in ischemic skin wounds.
Ascorbic acid, pantothenic acid and scars
This study aimed at testing human skin wound healing improvement by a 21-day supplementation of 1.0 g ascorbic acid (AA) and 0.2 g pantothenic acid (PA). 49 patients undergoing surgery for tattoos, by the successive resections procedure, entered a double-blind, prospective and randomized study. Tests performed on both skin and scars determined: hydroxyproline concentrations, number of fibroblasts, trace element contents and mechanical properties. In the 18 supplemented patients, it was shown that in skin (day 8) Fe increased (p < 0.05) and Mn decreased (p < 0.05); in scars (day 21), Cu (p = 0.07) and Mn (p < 0.01) decreased, and Mg (p < 0.05) increased; the mechanical properties of scars in group A were significantly correlated to their contents in Fe, Cu and Zn, whereas no correlation was shown in group B. In blood, AA increased after surgery with supplementation, whereas it decreased in controls. Although no major improvement of the would healing process could be documented in this study, our results suggest that the benefit of AA and PA supplementation could be due to the variations of the trace elements, as they are correlated to mechanical properties of the scars.
Vitamins enhance healing process
To study the effects of vitamins B5 and C on the healing process of colonic anastomoses, 3 groups of 20 rabbits were given daily either placebo (group A), or vitamin B5 (100 mg/kg: group B) or vitamin C (100 mg/kg: group C). After 8 days of supplementation, via a midline incision and under general anaesthesia, 2 colonic segments were removed, and the continuity was restored. On the 3rd post-operative day, the rabbits were killed and the anastomoses were removed. Mechanical properties of both normal colon and anastomoses were determined by using bursting pressure tests, number of burst anastomoses, fibroblast count, hydroxyproline concentration and determination by microanalysis of trace element content: Mg, P, S, Ca, Fe, Cu, Zn and Mn. Vitamin B5 (p = 0.03) and vitamin C (p less than 0.01) both decreased the number of burst anastomoses. Furthermore the required bursting pressure values were higher with vitamin C (p = 0.01) than in controls. Both vitamins restored normal Zn levels at the anastomotic site, whereas these levels decreased on the 3rd post-operative day during the normal healing process of colonic anastomosis. Moreover, vitamins B5 and C increased Fe, Cu and Mn levels, which are intimately all involved in collagen synthesis. Vitamins B5 and C enhance the colonic wound healing process in the rabbit, acting together in synergy in vivo as well as in vitro, as previously demonstrated.
Pantothenate and wound care
The effect of calcium D-pantothenate on the migration, proliferation and protein synthesis of human dermal fibroblasts from three different donors was investigated. The migration of cells into a wounded area was dose-dependently stimulated by Ca D-pantothenate. The number of cells that migrated across the edge of the wound increased from 32 +/- 7 cells/mm without Ca D-pantothenate to 76 +/- 2 cells/mm with 100 mg/ml Ca D-pantothenate. Moreover, the mean migration distance per cell increased from 0.23 +/- 0.05 mm to 0.33 +/- 0.02 mm. The mean migration speed was calculated to be 10.5 mm/hour without and 15 mm/hour with Ca D-pantothenate. Cell proliferation was also dose-dependently stimulated. The final cell densities were 1.2 to 1.6-fold higher in cultures containing 100 mg/ml Ca D-pantothenate. The protein synthesis was modulated, since two unidentified proteins were more strongly expressed in pantothenate supplemented cultures. In conclusion, Ca D-pantothenate accelerates the wound healing process by increasing the number of migrating cells, their distance and hence their speed. In addition, cell division is increased and the protein synthesis changed. These results suggest that higher quantities of pantothenate are locally required to enhance wound healing.
Blueberries, bilberry extract and fibrocystic mastopathy
The aim of this study was to further research into the therapeutic treatment of fibrocystic mastopathy. The study hypothesis included the clinical and instrumental control (echography) of patients with FCD before and after at least three months treatment with anthocyanosides. The protocol used was of the prospective and comparative type, whereas follow-up lasted three months. The study was performed in the outpatients' clinic of Breast Physiopathology and Echography organised within the ambit of the services of the Gynecology and Obstetrics Division. In this particular instance both echography and clinical examinations were performed at the same clinic. A total of 257 patients took part in the programme of which 35 were excluded since they failed to attend subsequent controls. Women were selected on the basis of absence of malignant disease and presence of clinical, echographic or mammographic symptoms of fibroso-cystic mastopathy. In addition, all women presented mastodynia which made therapy indispensable. The socio-demographic: characteristics of the population were polymorphous since women were resident not only in the area of USSL 36 but also other Italian provinces. The diameter of any lesions found was measured together with the thickness of the mammary gland. The thickness was always measured of QSE level both before and after treatment. The results were encouraging. There was a marked improvement in 75 patients, equivalent to 33.78%, symptoms were reduced in 61 women (27.47%) and disappeared in 14 (6.30%), whereas treatment had no effect in 72 cases (32.43%). In conclusion, echopalpation (clinical examination + echography) was extremely valuable in the study of these patients, especially if aged under 40. Moreover, therapy for three months in patients with mastodynia, consequent to fibrous mastopathy, was efficacious in reducing symptoms and mammary tension. At the same time, it is important to emphasize the absence of virtual absence of collateral effects to treatment. (Editors note: the European bilberry is Vaccinium myrtillus.)
Hepatic and kidney-type glutaminase
Glutamine is synthesized primarily in skeletal muscle, lungs, and adipose tissue. Plasma glutamine plays an important role as a carrier of nitrogen, carbon, and energy between organs and is used for hepatic urea synthesis, for renal ammoniagenesis, for gluconeogenesis in both liver and kidney, and as a major respiratory fuel for many cells. The catabolism of glutamine is initiated by either of two isoforms of the mitochondrial glutaminase. Liver-type glutaminase is expressed only in periportal hepatocytes of the postnatal liver, where it effectively couples ammonia production with urea synthesis. Kidney-type glutaminase is abundant in kidney, brain, intestine, fetal liver, lymphocytes, and transformed cells, where the resulting ammonia is released without further metabolism. The two isoenzymes have different structural and kinetic properties that contribute to their function and short-term regulation. Although there is a high degree of identity in amino acid sequences, the two glutaminases are the products of different but related genes. The two isoenzymes are also subject to long-term regulation. Hepatic glutaminase is increased during starvation, diabetes, and feeding a high-protein diet, whereas kidney-type glutaminase is increased only in kidney in response to metabolic acidosis. The adaptations in hepatic glutaminase are mediated by changes in the rate of transcription, whereas kidney-type glutaminase is regulated at a posttranscriptional level.
Neutrophils play an important role in host defense by phagocytosing and destroying invading bacteria. A recent investigation revealed that glutamine (Gln) augmented the in vitro bactericidal activity of neutrophils from burn patients. However, it is unclear whether Gln enhances the function of neutrophils in postoperative patients. This study was designed to investigate the effect of Gln on the in vitro Escherichia coli-killing activity of neutrophils from postoperative patients. Nine randomly selected patients were included in this study. On the morning of the first postoperative day, blood was drawn and neutrophils were isolated. Eight healthy volunteers served as controls. E. coli was opsonized with pooled normal serum. Neutrophils (5 x 10(6)), together with opsonized E. coli (5 x 10(5)), were incubated for 2 h at 37 degrees C in Hanks' balanced salt solution supplemented with 0, 100, 500, or 1000 nmol/mL of Gln. The bactericidal function of neutrophils was determined by counting the number of viable bacteria. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-8, and granulocyte elastase levels in the cell culture supernatant were measured. Plasma C-reactive protein (CRP), cortisol, and amino acids were also analyzed. The plasma concentration of Gln was significantly lower in the postoperative patients than in the controls. Following culture with patient neutrophils, the number of viable E. coli decreased by 26% as the in vitro Gln concentration was increased from 500 to 1000 nmol/mL (P < 0.01). We defined the Gln 1000/Gln 500 ratio of the number of viable bacteria as the number of viable E. coli at an in vitro Gln concentration of 1000 nmol/mL divided by the number of viable E. coli at an in vitro Gln concentration of 500 nmol/mL. A positive correlation was thus demonstrated between the plasma Gln level and the Gln 1000/Gln 500 ratio of the number of viable bacteria in the patients (r = 0.69, P = 0.04). This finding indicated that as plasma Gln fell, there was an enhancement of neutrophil E. coli-killing activity by neutrophils in in vitro tests when the Gln concentration was increased from 500 to 1000 nmol/mL. Gln supplementation caused no appreciable changes in TNF-alpha, IL-1 beta, IL-8, or granulocyte elastase levels in cell culture supernatants. A negative correlation was recognized between the patient plasma Gln level and the Gln 1000/Gln 500 ratio of the cell culture supernatant IL-8 level (r = -0.73, P = 0.025). In conclusion, Gln supplementation enhanced the in vitro bactericidal function of neutrophils from postoperative patients.
Glutathione suppresion of PGE2 synthesis
Reduced natural killer (NK) activity found in tumor-bearing hosts has been associated with high levels of prostaglandin E2 (PGE2) produced by monocytes in vitro. We have previously demonstrated a dependence of NK cell activity on glutamine (GLN) levels in vitro and in vivo. Further, glutathione (GSH) is antagonistic to PGE2 synthesis. We hypothesized that GLN, through increased GSH production, leads to decreased PGE2 synthesis and upregulation of NK cytotoxic activity. To test this, we examined the effects of oral GLN on GSH and PGE2 concentrations, NK activity and tumor growth in a rat breast cancer model. Starting on the day of MTF-7 tumor implantation 18 Fisher 344 rats were pair-fed chow and gavaged with 1 g/kg/day GLN (n = 9) or an isonitrogenous amount of Freamine (FA) (n = 9). Seven weeks after tumor implantation rats were sacrificed. Tumors were measured, weighed, and processed for tumor morphometrics. Spleens were removed, lymphocytes isolated and assayed for NK activity. Blood GLN, GSH, and PGE2 concentrations were measured. Over the 7-week study period tumor growth was decreased by approximately 40% in the GLN-supplemented group. This decrease in growth was associated with a 2.5 fold greater NK activity in the GLN-fed rats vs FA-fed rats. This correlated with a 25% rise in GSH concentration and a proportional decrease in PGE2 synthesis. Decreased tumor volume in rats fed GLN was not associated with changes in morphometrics. Oral GLN supplementation enhances NK activity resulting in decreased tumor growth. The enhanced NK activity seen with oral GLN supplementation in the tumor-bearing host is associated with GSH mediated suppression of PGE2 synthesis.
Glutamate and hypoxia-induced oxidative stress
High altitude stress leads to lipid peroxidation and free radical formation which results in cell membrane damage in organs and tissues, and associated mountain diseases. This paper discusses the changes in biochemical parameters and antibody response on feeding glutamate to male albino Sprague Dawley rats under hypoxic stress. Exposure of rats to simulated hypoxia at 7576 m, for 6 h daily for 5 consecutive days, in an animal decompression chamber at 32 +/- 2 degrees C resulted in an increase in plasma malondialdehyde level with a concomitant decrease in blood glutathione (reduced) level. Supplementation of glutamate orally at an optimal dose (27 mg/kg body weight) in male albino rats under hypoxia enhanced glutathione level and decreased malondialdehyde concentration significantly. Glutamate feeding improved total plasma protein and glucose levels under hypoxia. The activities of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) and the urea level remained elevated on glutamate supplementation under hypoxia. Glutamate supplementation increased the humoral response against sheep red blood cells (antibody titre). These results indicate a possible utility of glutamate in the amelioration of hypoxia-induced oxidative stress.
Differentiating agents have been used experimentally and clinically as an adjuvant in the treatment of cancer, but their role in chemoprevention is limited. We used 5% dimethylsulfoxide (DMSO), 1% and 4% methylsulfonylmethane (MSM), 0.3% N-methylformamide (NMF), and retinol acetate (RA) in the chemoprevention of rat mammary breast cancer. One hundred fifty 42-day-old Sprague-Dawley rats were randomized into six groups (control, RA, DMSO, 1% MSM, NMF, and 4% MSM) and received chemopreventive agents along with standard rat chow ad libitum. Eight days later, 15 mg of 7,12-dimethylbenzanthracene was given by oral gastric intubation. The animals were examined weekly for tumor incidence and size (biplanar analysis). Animals were followed up for 240 to 300 days. Tumor incidence was not statistically affected. Time to appearance (latency period) of both tumors and cancers were prolonged by NMF, DMSO, and 4% MSM. Doubling times of all cancers produced were prolonged by DMSO and RA. No group exhibited toxic reactions or significant weight loss. Polar solvents and differentiating agents, specifically NMF, DMSO, and 4% MSM, were effective in the chemoprevention of dimethylbenzanthracene-induced mammary cancers.
Polar solvents and colon cancer
To examine the effect of the polar solvents on 1,2-dimethylhydrazine (DMH)-induced colon cancer, 100 male Sprague-Dawley rats were randomly allocated to a control and three treatment groups. Treated animals received N-methylformamide (NMF), dimethylsulfoxide (DMSO), or methylsulfonylmethane (MSM) added to drinking water 1 week before carcinogen injections commenced and for the duration of the experiment. Primary tumors were detected by serial laparotomy under ether anesthesia performed at 2-month intervals and commencing after carcinogen injections had been completed. The average time to tumor onset was significantly delayed in rats receiving NMF and MSM (P = 0.0141 and 0.0398 respectively, Mantel-Haenszel test). In addition, fewer poorly differentiated tumors were noted in treatment groups. No weight loss or toxicity was observed. These findings demonstrate that the polar solvents significantly reduce the latent period to tumor onset in DMH-induced colon cancer and indicate the need to further investigate such compounds as chemopreventive agents.
Chondroitin sulfate's effects on osteoarthritis
OBJECTIVE: To assess the clinical efficacy of chondroitin sulfate (CS) in comparison with the nonsteroidal antiinflammatory drug (NSAID) diclofenac sodium (DS) in a medium/longterm clinical study in patients with knee osteoarthritis (OA). METHODS: This was a randomized, multicenter, double blind, double dummy study. 146 patients with knee OA were recruited into 2 groups. During the first month, patients in the NSAID group were treated with 3 x 50 mg DS tablets/day and 3 x 400 mg placebo (for CS) sachets; from Month 2 to Month 3, patients were given placebo sachets alone. In the CS group, patients were treated with 3 x 50 mg placebo (for diclofenac) tablets/day and 3 x 400 mg CS sachets/day during the first month; from Month 2 to Month 3, these patients received only CS sachets. Both groups were treated with 3 x 400 mg placebo sachets from Month 4 to Month 6. Clinical efficacy was evaluated by assessing the Lequesne Index, spontaneous pain (using the Huskisson visual analog scale), pain on load (using a 4 point ordinal scale), and paracetamol consumption. RESULTS: Patients treated with the NSAID showed prompt and plain reduction of clinical symptoms, which, however, reappeared after the end of treatment; in the CS group, the therapeutic response appeared later in time but lasted for up to 3 months after the end of treatment. CONCLUSION: CS seems to have slow but gradually increasing clinical activity in OA; these benefits last for a long period after the end of treatment.
Distribution of radioactivity and chondroitin sulfate
After the administration of tritiated chondroitin sulfate (CS) by oral and intramuscular route, the distribution of radioactivity was investigated in two opportunist omnivorous animals, namely the rat and the dog. More than 70% of the orally administered radioactivity was absorbed. Independently of the administration route, radioactivity was mainly excreted through the urine. Plasma levels showed a rapid increase after oral administration, followed by a large plateau with a maximum at the 14th and 28th h in the rat and in the dog, respectively. A tropism of the radioactivity was observed towards glycosaminoglycan-rich tissues, such as joint cartilage. The analysis of the molecular weight of the radioactive material showed that compounds with a molecular weight corresponding to those of CS, poly-, oligo- and monosaccharides as well as of tritiated water, were present in the plasma, urine, synovial fluid and cartilage. The level of radioactive low molecular weight material, derived from the metabolism of CS and from the exchange reaction, increased with the time after administration. The high molecular weight fraction represented at least 10% of the orally administered CS.