Nitric oxide (NO), a gaseous free radical, is a critical mediator of normal tissue repair.1 A deficiency in NO is associated with diabetes-impaired wound healing.2-4 Angiogenesis,5 granulation tissue formation,6 epidermal migration,7 and collagen deposition8 are all significant healing processes that are regulated by optimal NO bioactivity. Experimental studies also document a biphasic effect of NO on the wound keratinocyte, with cellular proliferation or cytostasis documented as a result of low (basal) or high (toxic) NO tissue concentrations, respectively.9-11
This article presents a case of chronic bilateral, medial ankle venous ulcerations in a patient with type 2 diabetes and an untreated, elevated serum homocysteine (Hcy) level. The patient's wounds had failed to heal after treatment with topical human fibroblast-derived dermal substitute (Dermagraft). Following the reduction of elevated Hcy and reapplication of Dermagraft, however, both wounds healed after 4 weeks of treatment. This case documents an inverse relationship between elevated serum Hcy and decreased wound NO bioactivity by measuring nitrate and nitrite (NOx; the stable oxidation products of NO) in the wound fluid of the treated ulcers.
In experimental and clinical wound healing research, NOx has been used exclusively as a reliable surrogate marker for NO.1-4,6,8 In all cases, NOx levels have been highly sensitive to conditions or factors that reduce NO production and impair normal wound healing, such as diabetes,12 protein-calorie malnutrition,13 cutaneous irradiation,14 steroid therapy,15 and metabolic inhibition of NO synthesis.16 In these cases, decreased wound fluid NOx and impaired wound closure were associated with decreased collagen accumulation,17 wound tensile strength, type I and III collagen gene expression,18 vascular endothelial growth factor expression, granulation tissue formation, and wound microvascular perfusion.19
Although Hcy has been documented to antagonize NO bioactivity, its association with impaired wound healing has not been previously documented. This case presents clinical evidence suggesting that medical treatment of an elevated Hcy level successfully reverses Hcy-mediated inhibition of wound NO bioactivity associated with impaired wound healing.
History and Examination
Besides bilateral medial ankle venous ulcerations, the patient, a 79-year-old white male, had well-controlled type 2 diabetes mellitus (A1C, 6.8%), hypertensive cardiovascular disease, chronic bilateral venous insufficiency, peripheral vascular disease, elevated fasting serum Hcy (14.9 micromol/L), and lower extremity neuropathy. His medications included insulin (Novolin [70/30]), furosemide, atenolol, potassium chloride, and aspirin. The patient was 6′8″ tall and weighed 228 pounds, with a body mass index of 25.75. A nonsmoker, he was physically active and well nourished, with normal prealbumin and albumin levels of 185 mg/L and 3.8 g/dL, respectively.
At the time of evaluation, the patient's ulcers had remained unhealed for 24 months. He had received ongoing compression therapy, initially with a short-stretch bandaging technique to reduce edema, then with application of compression hose providing 20 to 30 mm Hg of compression. Wound care consisted of normal saline irrigation, application of gentamicin ointment, and wet-to-wet dressing technique. Before evaluation, the patient received adjunctive hyperbaric oxygen treatment (20 treatments; 2 atmosphere absolute × 90 minutes) and treatment with topical rhPDGF-B (becaplermin, REGRANEX Gel) growth factor therapy for 20 weeks, with neither therapy promoting successful wound healing. The ulcers showed no signs of infection, and cultures were negative for pathogens. Pulses were palpable on both ankles, and excisional pathology of the wounds demonstrated no malignant changes. The right medial ankle ulcer was 4.4 cm2 and the left medial ankle ulcer was 1.1 cm2.
The ulcers were shallow (approximately 2 mm deep), and full thickness, with anemic granulation tissue at the base of each ulcer without exposed bone or tendon. Wound fluid NOx was used as a surrogate biomarker for NO bioactivity2,6,8, and was measured with nitrate-free filter paper left on the ulcer for 24 hours. Wound fluid NOx of the right ankle ulcer was 1.5 micromoles, which was significantly lower (P < .05) than the mean wound fluid NOx (13.9 ± 2.3 micromoles; n = 13)for lower extremity ulcers with normal healing responses and normal serum Hcy at the authors' wound center.20
Dermagraft, a bioengineered fibroblast-based growth factor/cytokine topical wound treatment, was applied with compression therapy to both medial ankle ulcers for 8 consecutive weeks to stimulate wound angiogenesis, granulation tissue formation, and wound closure. After 2 weeks, the right ankle ulcer area decreased by 50% (2.2 cm2); it did not further reduce in size with subsequent treatment. The left ankle ulcer increased in area following Dermagraft treatment and was 40% larger by the fifth week of treatment. The left ankle ulcer area returned to its baseline area (1.1 cm2) by the eighth week of treatment.
Following 8 weeks of treatment, the bilateral ulcer areas did not significantly change for the next 2 months. During this time, the ulcers were treated with a topical hydrocolloid dressing and compression therapy.
Wound fluid NOx measurements for the right ankle ulcer were repeated 2, 4, 6, and 8 weeks after beginning Dermagraft treatment. Wound fluid NOx increased to a maximum of 2.6 micromoles after 4 weeks of Dermagraft treatment but remained significantly lower than normal values. Following this treatment course, wound fluid NOx levels returned to near-baseline values (1.5 micromoles) at the completion of 8 weeks of Dermagraft treatment.
During the 2 months following Dermagraft treatment, the patient was monitored at the authors' wound center. He showed no intercurrent illness or changes in glycemic control, weight, or nutritional status. Multivitamin therapy21 was prescribed for the treatment of his elevated serum Hcy (14.9 micromol/L; normal, <11.4 micromol/L). The multivitamin (Foltx) was administered twice a day and consisted of folic acid (5 mg), vitamin B6 (50 mg), and vitamin B12 (2 mg). After 3 weeks of treatment, the patient's fasting serum Hcy was lowered to 11.1 micromol/L.
A repeat wound fluid NOx measurement of the right ankle ulcer showed an increase to 6.5 micromoles. The right ankle ulcer area was 2 cm2 and the left ankle ulcer area was 1.5 cm2, with both granulating wound surfaces appearing more robust and vascular in appearance. Dermagraft was now reapplied to both ankle ulcers. At the end of 4 weeks of this second round of treatment with Dermagraft and compression therapy, both ankle ulcers were completely healed.
The patient continues on folic acid, B6 and B12 supplements, and compression therapy. His ankle ulcers have remained completely healed for the past 6 months.
Following treatment of this patient, the authors documented the incidence of untreated, elevated Hcy in their wound healing center population. During a 6-month period, 138 patients receiving treatment for chronic, nonhealing lower-extremity ulcers were evaluated. In this group, 50% had untreated elevated fasting serum Hcy; 69% of patients with diabetes and neuropathic ulcers had an elevated fasting Hcy compared with 47% of nondiabetic patients with lower-extremity ulcers. All patients with an elevated serum Hcy level were scheduled for treatment with folic acid and vitamins B6 and B12.
At this time a randomized, controlled trial evaluating the association between elevated Hcy, wound NO bioactivity, and wound fluid NOx is under way.
This case report documents an association between the successful use of Dermagraft to treat a chronic lower-extremity ulcer and the use of medical therapy with folic acid and vitamins B6 and B12 to reverse an elevated serum Hcy level. Although confirmation of a direct causal inference between elevated Hcy and impaired wound healing cannot be established from this single case, Hcy-mediated inhibition of wound NO bioactivity by measurement of wound fluid NOx may implicate elevated Hcy as a risk factor for patients receiving treatment for a chronic lower-extremity ulcer.
Hcy is a nonessential amino acid that antagonizes NO production via multiple pathways, including inhibition of arginine transport,22 inhibition of the breakdown of the NO inhibitor asymmetric dimethylarginine (ADMA),23 and pro-oxidant behavior.24
A chronic, elevated serum Hcy level is considered a significant risk factor for cardiovascular disease, such as atherosclerosis, hypertension, cerebrovascular accident, myocardial infarction, and pulmonary embolism. Elevated Hcy levels are usually caused by inborn errors of homocysteine metabolism, deficiencies of folic acid and vitamin B6 and B12,diseases such as chronic renal failure, and drugs interfering with homocysteine metabolism.25 Other factors such as cigarette smoking, excessive caffeine consumption, and spinal cord injury have also been associated with Hcy elevation.26-28
Experimental studies suggest that an elevated serum Hcy level may act to inhibit wound repair by occupying the fibronectin domain of fibrin during provisional wound matrix formation.29 These studies demonstrated that Hcy binds to fibronectin by up to 62% and is increased with a rising Hcy concentration. Hcy has also been observed to inhibit angiogenesis30 and may impair collagen metabolism.31 The competitive inhibition of the fibronectin-binding site for fibrin, inhibition of NO production, and angiogenesis and wound matrix formation may represent mechanisms by which elevated Hcy impairs wound healing Figure 1.
Because an elevated Hcy level may be a common occurrence in patients with chronic wounds, the evaluation process for these patients may need to be revised. This case also illustrates the potential value of wound fluid NOx measurement as a risk factor assessment method and noninvasive diagnostic tool for clinicians treating patients with chronic ulcers. If confirmed with additional studies, these relationships may provide valuable information about a novel clinical aspect of Hcy management associated with impaired wound healing.
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