Homocysteine– A Stealth Mediator of Impaired Wound Healing: A Preliminary Study

Nitric oxide (NO), a gaseous free radical, is a critical mediator of normal tissue repair.¹ Angiogenesis,² granulation tissue formation,³ epidermal migration,⁴ and collagen deposition5 are all significant wound repair processes that are regulated by NO bioactivity. General somatic and wound NO bioactivity may be evaluated by measuring nitrate and nitrite (NOx, the stable oxidation products of NO) from plasma and urine and from wound fluid NOx, respectively. In experimental and clinical wound healing research, NOx has been used extensively as a reliable surrogate marker for NO.^1,3,5–8 In all cases, NOx determinations have been highly sensitive to conditions or factors that reduce NO production and impair normal wound healing, such as diabetes,⁹ protein-calorie malnutrition,¹⁰ cutaneous irradiation,¹¹ steroid therapy,¹² and metabolic inhibition of NO synthesis.¹³ In these cases, decreased wound fluid NOx and impaired wound closure were associated with decreased collagen accumulation,¹⁴ wound tensile strength, type I and III collagen gene expression,¹⁵ vascular endothelial growth factor (VEGF) expression, granulation tissue formation, and wound microvascular perfusion.¹⁶
Elevated homocysteine (Hcy) is widely accepted as a novel risk factor associated with atherosclerotic cardiovascular disease (CVD) in the coronary, cerebral, and peripheral vascular beds.¹⁷ Homocysteine also is believed to antagonize NO bioactivity by multiple pathways but has not been identified as a risk factor for wound repair. This preliminary study was designed to prospectively document the relationship between impaired wound healing, wound NO bioactivity evaluated by wound fluid NOx measurement, and elevated Hcy while using topical human fibroblast-derived dermal substitute (Dermagraft® Human Fibroblast-Derived Dermal Substitute, Smith & Nephew Inc., Largo, Fla) for the treatment of chronic lower-extremity ulcers.

Methods and Materials

Dermal substitute therapy. During the authors’ observations, 12 consecutive patients who were scheduled for topical human fibroblast-derived dermal substitute therapy for lower-extremity ulcers (LEUs) at the Retreat Hospital Wound Healing Center were selected for study. The product is a tissue-engineered human (neonatal) fibroblast-derived dermal substitute designed as an interactive therapy for the treatment of full-thickness diabetic foot ulcers greater than 6 weeks duration. When applied to the wound bed, the dermal substitute delivers a diverse elaboration of growth factors, cytokines, matrix proteins, and glycosaminoglycans. These components are found in healthy human dermis and have been demonstrated to encourage dermal matrix development, angiogenesis, epithelial cell migration, and wound closure. For ulcer treatment, patients received 8 weekly applications of dermal substitute to the debrided wound bed. Patients with signs of soft tissue infection or osteomyelitis were excluded from treatment. Patients whose ulcers displayed enhancements of wound healing parameters and a progressive demonstration of wound granulation tissue deposition, increased wound vascularity, and decreasing wound area and volumes following the initiation of dermal substitute therapy were considered “responders” to dermal substitute therapy. Patients whose wounds displayed minimal or no improvements with wound healing parameters were considered “non-responders” to dermal substitute therapy. Investigational review board approval and informed patient consent were obtained prior to the performance of these observations. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.
Wound fluid NOx determinations. During this study, baseline (pre-treatment) wound fluid NOx determinations were obtained from patients receiving dermal substitute therapy. Additionally, 4 study patients were selected for serial evaluation of wound fluid NOx and wound area determinations following dermal substitute therapy. Two of these patients (1 responder and 1 non-responder) are presented with illustrations of the serial profile of wound fluid NOx values and wound area measurements following treatment (Table 1, WFN patients). For this demonstration, wound fluid NOx determinations were made prior to dermal substitute treatment (baseline value) and at weeks 2, 4, 6, and 8 of dermal substitute treatment. Wound fluid from the study ulcer was collected with nitrate-free filter paper. After placing the filter paper on the wound for 24 hours with the routine dressing (in a silver- [Ag+] free environment), the wound fluid was eluted from the filter paper and the wound fluid NOx was determined by using the Griess assay.¹⁸ Wound fluid NOx samples were stored at -70˚C until the assay was performed. Laboratory personnel performing the assays were blinded to the identification of study subjects.
Wound area measurements. Measurements were performed directly on the wound (multiple measurements with averaged data). Estimations were then made of the wound area (cm²) for comparative analysis. Wound area measurements were performed immediately prior to initial dermal substitute application (baseline measurement) and at weeks 2, 4, 6, and 8 of therapy.
Serum Hcy determinations. Serum Hcy determinations were not performed until the completion of dermal substitute therapy for each patient. For this reason, no patients with untreated, elevated Hcy received any medical therapy for this condition during the study. Fasting serum Hcy samples were collected by the laboratory services at HCA Retreat Hospital and processed by Quest Diagnostics (Chantilly, Va). During this study, cardiovascular standards for serum Hcy were used to determine whether levels were elevated or within normal limits. These cardiovascular parameters were normal values < 11.4 mmol/L for adult men and normal values < 10.4 mmol/L for adult women. All determinations were made from fasting blood samples, which were promptly prepared following lab draw.
Statistical analysis. Statistical analysis of wound area measurements and wound fluid NOx was performed by 1-way analysis of variance (ANOVA) followed by Dunn’s post-test or paired t tests, as appropriate, with GraphPad Prism® (GraphPad Software, Inc., San Diego, Calif). The level of significance was taken as P < 0.05, and results are expressed as mean ± standard error (SE).

Results

Following dermal substitute therapy, all 12 patients receiving treatment were classified as either “responders” or “non-responders” to the 8-week treatment (Table 1). Demographic parameters for study patient comparisons for wound healing included the initial wound area prior to treatment (baseline ulcer area), the wound area following 2 weeks of dermal substitute therapy (ulcer area at 2 weeks), and the corresponding percentage reduction in wound area for that patient (% reduction ulcer area) (Table 1). Homocysteine determinations were recorded following the completion of treatment and were listed as either normal or elevated according to the cardiovascular parameters previously outlined (Table 2). The number of dermal substitute patients experiencing complete wound closure within 12 weeks of the completion of dermal substitute treatment was also recorded (Table 2). Patients responding to dermal substitute were observed with early wound contraction, robust granulation tissue formation, epidermal migration, and re-epithelization of the wound. Patients not responding to dermal substitute treatment were generally observed with a decreased rate of wound contraction, poor granulation tissue formation, and poor clinical evidence of epidermal migration or re-epithelization. For the entire group of 12 patients, 6 were classified as responders and the remaining 6 were classified as non-responders (Tables 1 and 2).
There were no significant differences between the baseline wound areas of responding and non-responding patients (Table 2). However, responding patients demonstrated a significantly greater rate of wound area reduction at 2 weeks as compared to the non-responding patients. All responding patients were observed with normal serum homocysteine levels, while 83% (5/6) of the non-responding patients were observed with elevated serum homocysteine levels. In the responder group, 67% of the patients displayed complete healing of their wounds within 12 weeks of the completion of dermal substitute treatments. None of the patients in the non-responder group were observed with wound closure during this same period. Mean wound fluid NOx determinations for the patients sampled from each group were obtained for the baseline measurement prior to dermal substitute therapy (Table 2). Prior to dermal substitute applications (baseline values), the mean wound fluid NOx value in micromoles (mmol/L ± SE) for the responders group was significantly elevated at 12.98 (± 1.73) as compared to the mean wound fluid NOx value of the non-responders group at 3.50 (± 1.13).
Mean wound fluid NOx value for the responder group was not significantly different from the mean wound fluid NOx (13.9 ± 2.3 mmol/L; n = 13) for lower-extremity ulcers with normal healing responses and normal serum Hcy at the authors’ wound center.¹⁹ However, the non-responder mean wound fluid NOx value is significantly lower than the mean wound fluid NOx value associated with normal wound healing and normal serum Hcy values.
Wound fluid NOx and wound area were correlated following dermal substitute treatment for lower-extremity ulcers for a responding and non-responding diabetic lower-extremity ulcer patient in Figures 1 and 2, respectively.
The dermal substitute responder was a 49-year-old woman with type II diabetes (HgbA1C 8.8%) and a history of rheumatoid arthritis, coronary artery disease, a plantar diabetic neuropathic ulcer, and diabetic retinopathy (Table 1, Patient #2). This patient was observed with a normal serum Hcy at the completion of the study. The baseline wound fluid NOx of the plantar neuropathic ulcer was 12.0 mmol/L, and the baseline wound area was 3.8 cm². Following 2 weeks of dermal substitute treatment, wound fluid NOx had increased maximally to 18 mmol/L, which correlated with a 49% reduction in the area of the neuropathic ulcer. Following the second week of treatment, wound fluid NOx levels decreased along with wound area until the end of the eighth week of treatment when the wound had healed completely (Figure 1). All monitored responder patients displayed a similar pattern of maximal wound fluid NOx elevation at 2 weeks following dermal substitute application. These increases ranged from 130% to 150% above baseline values for wound fluid NOx.
The dermal substitute non-responder was a 79-year-old man with type II diabetes (HgbA1C 6.8%) and a history of hypertension, coronary artery disease, spinal stenosis, status/post left femoral-popliteal bypass, and bilateral medial ankle venous stasis ulcerations (Table 1, Patient #5). This patient had an untreated, elevated serum Hcy of 14.9 mmol/L measured at the end of the study. The baseline wound fluid NOx (right ankle sample only) was 1.47 mmol/L. The initial area of the right ankle ulcer was 4.4 cm²; the initial area of the left ankle ulcer was 1.1 cm² (Figure 2). Wound fluid NOx increased gradually following dermal substitute application and was maximally elevated at 4 weeks (2.6 mmol/L). During this period, the right ankle ulcer area was reduced by more than 60%; however, the left ankle ulcer area during this same time increased by about 50% (2.2 cm²). By the end of the eighth week of dermal substitute treatment, the right ankle ulcer area was unchanged from the area at 4 weeks; however, the left ankle ulcer area during the last 4 weeks of treatment declined and had returned to near baseline value by the end of the eighth week of treatment. The wound fluid NOx levels during the last 4 weeks of dermal substitute treatment declined and had returned to near baseline values (1.5 mmol/L) by the end of the eighth week of treatment.

Case Report

Following the completion of the initial trial of dermal substitute treatments on the 12 study patients, a decision was made to attempt to reverse the impaired wound healing of the selected non-responder patient (Patient #5, Table 1) by medical treatment of his elevated serum Hcy while continuing to monitor wound fluid NOx. The non-responder was a 79-year-old male with diabetes who was 6'8" tall, weighed 228 lb, and had a body mass index of 25.75. His medications included insulin (Novolin® 70/30, Novo Nordisk Pharmaceuticals, Inc., Princeton, NJ), furosemide, atenolol, potassium chloride, and aspirin. He was a non-smoker, physically active, and well nourished with normal pre-albumin and albumin levels of 185 mg/L and 3.8 gm/dL, respectively.
Prior to the initial dermal substitute treatment, the patient’s ulcers had remained unhealed for 24 months. During this time, the patient had received ongoing compression therapy, wound care, adjunctive hyperbaric oxygen treatment (20 treatments at 2 ATA for 90 minutes), and topical rhPDGF-B (Regranex®, Johnson & Johnson Wound Management, Somerville, NJ) growth factor therapy (20 weeks). The ulcers showed no signs of infection, and cultures were negative for pathogens. Pulses were palpable on both ankles. Excisional pathology of the wounds demonstrated no malignant changes.
During the next 2 months following dermal substitute treatment, the non-responder patient was followed in the wound center and was observed with no intercurrent illness and no change in glycemic control, weight, or nutritional status. The patient then began receiving multivitamin therapy²⁰ for the treatment of his elevated serum Hcy (14.9 mmol/L). This treatment consisted of 2.5 mg folic acid, 25 mg pyridoxine, and 2 mg cyanocobalamin (Foltx®, Pamlab, LLC, Covington, La) administered twice a day. After 3 weeks of treatment, the fasting serum Hcy was lowered to 11.1 mmol/L (within normal limits: < 11.4 mmol/L). The WFNOx measurement of the right ankle ulcer was now repeated and had increased to 6.5 mmol/L. Ulcer examination at this time documented a left ankle ulcer area of 1.5 cm² and a right ankle ulcer area of 2 cm² with both granulating wound surfaces appearing more robust and vascular in appearance. Both ankle ulcers were now retreated with dermal substitute. After 4 weeks of retreatment with dermal substitute and compression, both ankle ulcers were completely healed (Figures 3 and 4).
For the next 6 months following ulcer closure, the patient’s serum Hcy was monitored regularly and demonstrated moderate fluctuations (11.1–13.4 mmol/L) that were associated with marginal inflammation. Approximately 8 months after closure, partial recurrence at the ulcer site was evident. At this time, the patient’s medical treatment for elevated serum Hcy was changed to a combination treatment of 2.8 mg L-methylfolate, 25 mg pyridoxal 5’-phosphate, and 2 mg methylcobalamin (Metanx™, Pamlab, LLC) administered twice daily. With this change in therapy, the patient’s serum Hcy decreased to 9.7 mmol/L and did not demonstrate elevations above this value. Also, approximately 1 month after this change in treatment, the wounds healed completely and no longer displayed inflammatory changes (Figure 5). He remained healed at 9 months follow-up.
Following the treatment of this patient, the authors documented the incidence of untreated, elevated Hcy in the wound healing center population. During a 6-month survey, the authors evaluated 138 consecutive patients receiving treatment for chronic, nonhealing LEUs. In this group, 50% were observed with untreated, elevated fasting serum Hcy. Furthermore, patients with diabetes and neuropathic ulcers had a 63% incidence of elevated fasting Hcy; LEU patients without diabetes had a 47% incidence of elevated fasting Hcy.

Discussion

The results of this preliminary clinical study suggest a correlation exists between elevated serum Hcy and impaired chronic wound healing with patients receiving dermal substitute therapy. Furthermore, the preliminary data indicate that elevated serum Hcy may also be correlated with significantly decreased wound NO bioactivity as determined by wound fluid NOx assay, and left untreated, elevated Hcy may become common among patients with chronic wounds (50% incidence). Additionally, the observations of a single case report suggest that successful treatment of elevated Hcy in a patient with impaired wound healing may promote the restoration of normal wound healing.
Nitric oxide, a key mediator of cutaneous physiology, is formed by the enzymatic combination of molecular oxygen and the semi-essential amino acid L-arginine. Nitric oxide provides cellular signaling by activation of its target molecule, guanylate cyclase, which elevates intracellular concentrations of cyclic guanosine monophosphate (cGMP).²¹ Increased cGMP causes vascular smooth muscle relaxation, which constitutes a significant mechanism of homeostasis for microcirculation, and modulates the cardiovascular response to vasoconstrictors, cytokines, and endotoxin. Nitric oxide may alter key enzymes, affecting subcellular systems, the Krebs cycle, or RNA/DNA synthesis. This activity is performed without the need for signal transduction. Nitric oxide crosses cell membranes without mediation of channels or receptors—it diffuses across cellular membranes isotropically.²¹ Because of its high diffusion coefficient, short half-life (approximately 5 seconds), and prompt decomposition, NO is ideal because it acts as a cellular signal for wound repair. Nitric oxide is generated by 3 isoforms of nitric oxide synthase (NOS) that metabolize L-arginine and molecular oxygen to citrulline and NO.²² Two of the 3 isoforms are constitutive enzyme systems (cNOS) that are described in neuronal cells (nNOS) and endothelial cells (eNOS). With these enzymes, increased levels of intracellular calcium activate the cNOS via calmodulin. The calcium-dependent cNOS systems produce low (picomolar) quantities of NO. The third system is the inducible isoform (iNOS), which is calcium independent. Expression of iNOS is controlled by tissue-specific stimuli, such as inflammatory cytokines or exogenous materials, ie, bacterial lipopolysaccharide (LPS). Once induced, production of NO within tissue can increase as much as 1,000-fold, thereby producing an environment that is toxic to invading microorganisms. Currently, it appears that the cNOS enzymes are involved in maintaining skin homeostasis and providing regulatory function.²² The iNOS enzymes appear to be mainly associated with inflammatory and immune responses that are also implicated in certain skin diseases. In human skin, keratinocytes, fibroblasts, and endothelial cells possess both the cNOS and iNOS isoforms. The wound macrophage and keratinocyte possess the iNOS isoform.²³ Epithelial migration,⁴ wound angiogenesis,² and granulation tissue formation³ are primarily mediated by the activation and upregulation of the iNOS isoform.
The major metabolic pathway for NO is through nitrate (NO3–) and nitrite (NO2–), collectively termed NOx, which are stable metabolites within tissue, plasma, and urine.²¹ Tracer studies in humans have demonstrated that perhaps 50% of the total body NOx originates from the NO synthesis substrate, L-arginine, although this percentage will vary with the dietary intake of NOx.^24,25 Fasting plasma and urine samples allow clinicians to use variations in NOx values as a means of evaluating changes in NO production and bioactivity.²⁶
Ongoing experimental and clinical wound healing studies have now clearly established NO as a critical mediator of normal tissue repair.¹ Angiogenesis, granulation tissue formation, epidermal migration, collagen deposition, and microvascular homeostasis are significant vulnerary processes, critical to normal wound repair, that are regulated by NO production and bioactivity.^1–4,13 Functional recovery of cutaneous vascular beds sustaining ischemia-reperfusion injury²⁷ and increased local random tissue flap survival²⁸ have also been linked to the NO-mediated enhancement of vascular (endothelial) modulation in experimental models. Optimal NO activity is required for the full expression and receptor upregulation of VEGF and platelet-derived growth factor (PDGF).^29,30 A deficiency in NO bioactivity is associated with diabetes-impaired wound healing.⁶ Successful recombinant topical PDGF-BB (becaplermin; Regranex) therapy for chronic LEU patients with diabetes may also be dependent upon optimal NO production for successful wound treatment.⁷
A distinctive feature of the wound fluid NOx profile documented for the responder patient highlighted in this article is the early supernormal elevation of wound fluid NOx that is recorded after 2 weeks of dermal substitute treatment (Figure 1). At this time, wound fluid NOx increased 130% to 150% above the baseline value where it was significantly greater than the mean wound fluid NOx value associated with normal wound healing. This phenomenon was not observed with the non-responding patient. For the non-responding patient, the baseline wound fluid NOx value (1.47 mmol/L) was significantly lower than the mean wound fluid NOx value associated with normal wound healing. This relationship was unchanged when maximal elevation in wound fluid NOx was observed (2.61 mmol/L) in the non-responding patient at 4 weeks after treatment. Maximal wound fluid NOx levels at 2 weeks following dermal substitute application correlated with significant reductions in wound areas for the responding patients as compared to wound area changes for the non-responding patients. Wound contraction and epidermal migration are the mechanisms responsible for area reduction in the actively healing wound. In experimental studies, endogenous NO production is a prerequisite for epidermal cell migration and may function to switch the epithelial cell from the stationary to the locomoting phenotype.⁴ The vectorial effect of wound NO in these studies was considered a critical factor for the spatial and temporal coordination of locomoting epithelial cells during wound closure. Epithelial cell migration in these studies was associated with an initial transient release of NO with inducible NOS having a high abundance at the edges of the epithelial wounds.⁴ These experimental findings appear to support the documented correlation between significantly decreased wound areas and early, transiently increased NO activity (wound fluid NOx) as was the case with the responding dermal substitute patients.
Howdieshell³¹ documented a pattern of wound fluid NOx enhancement associated with a normal wound healing response that is similar to that observed with the responding dermal substitute patient. Using an experimental porcine ventral hernia wound model, Howdieshell measured wound fluid NOx, wound vascular perfusion and VEGF, and transforming growth factor-b (TGF-b) expression during normal wound repair and following the inhibition of iNOS. In this study, the maximal elevation of wound fluid NOx occurred on Day 11 of the study and was preceded by increased basal and heat provoked wound perfusion and increased thickness of healthy granulation tissue. This maximal elevation in wound fluid NOx was significantly greater than pre-injury values and correlated with significant elevations in the concentrations of VEGF and TGF-β from the granulating wound surface. After pharmacologic inhibition of iNOS, a significant reduction in wound NO bioactivity and wound fluid NOx was associated with decreased granulation tissue formation, VEGF expression, and impaired wound healing. Despite the limited clinical observations made in the present study, the authors believe that the supernormal elevations in wound fluid NOx associated with a normal wound healing response observed by Howdieshell are supportive of the present clinical findings. The present findings have demonstrated similar patterns of maximally increased wound fluid NOx (at about 2 weeks) that were associated with a favorable response for wound closure. Furthermore, given the identification of a normal wound fluid NOx threshold for normal healing established from the authors’ center, these observations suggest that baseline wound fluid NOx determinations may be predictive of successful outcome for the patient receiving dermal substitute treatment.
Homocysteine is an intermediate sulfur-containing amino acid formed during the intracellular metabolism of methionine, an essential amino acid supplied by dietary proteins.¹⁷ The factors responsible for hyperhomocysteinemia include inborn errors of homocysteine metabolism, drugs that interfere with homocysteine metabolism, deficiencies of folic acid, vitamin B6 (pyridoxal 5’-phosphate) or vitamin B12 (methylcobalamin), and certain disease states, such as chronic renal failure, that increase total plasma homocysteine levels.¹⁷ The prevalence of hyperhomocysteinemia for the general population and for individuals with metabolic syndrome or type II diabetes mellitus is estimated between 5% and 7%.³² These figures underscore the significance of the documented incidence of elevated Hcy in the authors’ chronic wound population where 50% of these patients and 63% of patients with diabetes mellitus and neuropathic LEU were observed with untreated, elevated Hcy levels.
The inborn errors of homocysteine metabolism generally imply a genetic deficiency of cystathionine b-synthase; N5, N10-methylenetetrahydrofolate reductase (MTHFR) C677T; and methionine synthase (MTR).³³ Environmental factors that cause elevated Hcy include nutrition, lifestyle, physiological conditions, drugs, and some diseases. Deficiencies of folate, vitamin B12, and vitamin B6 are the most frequent nutritional defects causing hyperhomocysteinemia.³³ Among lifestyle factors, cigarette smoking, high alcohol intake, and coffee consumption are the major factors implicated, possibly via impaired folate or vitamin B6 function.³³ Aging and menopause are physiological conditions associated with elevated Hcy that may relate to a physiological decline in renal function with age. Drugs, such as methotrexate, the immunosuppressive cyclosporine, antidiabetic (metformin), anticonvulsant (carbamazepine) or hypolipidemic drugs (cholestyramine, niacin), may lead to elevated Hcy due to folate antagonism or by inhibition of vitamin B6 function. Also, disease states, such as chronic renal failure, hypothyroidism, systemic lupus erythematosus, and several cancers, can enhance fasting plasma Hcy levels, probably by low vitamin status, impaired enzyme function, or increased Hcy export from proliferating cells.³³ Spinal cord injury has also been associated with an increased incidence of elevated serum Hcy.³⁴
Multivitamin therapy, including folic acid, vitamin B6, and vitamin B12 in elevated doses, has been referenced as an acceptable treatment for elevated Hcy.²⁰ Folic acid normally requires 4 separate biochemical reactions for its conversion into L-methylfolate, the bioavailable form of folate directly involved in Hcy metabolism. These steps include the conversion of folic acid to dihydrofolate (DHF) by dihydrofolate reductase enzyme (DHFR); the metabolism of DHF into tetrahydrofolate (THF) by DHFR; the metabolism of THF into 5,10-methylene-THF; and finally the conversion of 5,10-methylene-THF into L-methylfolate by the methyltetrahydrofolate reductase enzyme (MTHFR).³² A polymorphism of MTHFR (C677T) is common in most populations with a homozygosity rate of 10%–15% that is associated with moderate hyperhomocysteinemia, especially in the context of marginal folate intake.³⁴ Overall, 40%–50% of the population exhibit a genetic polymorphism in which folic acid is incompletely converted to the active isomer of folate for homocysteine metabolism.^35–38
The treatment regimen for elevated Hcy has been improved with the formulation of L-methylfolate. L-methylfolate, or (6S)-5-methyltetrahydrofolate (5-MTHF), is the primary biologically active isomer of folate and the active form of folate.³⁹ It is also the form that is transported across membranes into peripheral tissues, particularly across the blood brain barrier. By substituting L-methylfolate for folic acid in elevated Hcy treatment, the 4 separate biochemical reactions for folic acid conversion are bypassed and the L-methylfolate is immediately available for Hcy metabolism. Problems related to folate absorption associated with inborn errors of folic acid metabolism are significantly bypassed. As with folic acid treatment for elevated Hcy, L-methylfolate must be combined with pyridoxal 5’-phosphate and methylcobalamin, as these co-factors are important in preventing pernicious anemia and in maintaining neurofunctions, including myelinogenesis, nerve repair, and nerve regeneration.⁴⁰
L-methylfolate administration has proven superior to folic acid therapy and is 7 times more bioavailable than folic acid with a significant superiority (3X) in lowering serum homocysteine.³⁶ The superior homocysteine lowering effect of L-methylfolate was demonstrated in the present study as it substantially lowered patient serum Hcy from 11.3 mmol/L with folic acid therapy to 9.7 mmol/L with L-methylfolate treatment and promoted the recovery and maintenance of complete healing of the treated ankle ulcerations discussed in the case report (Figure 5). For these reasons, the authors recommend administration of the combination therapy of L-methylfolate, pyridoxal 5’-phosphate, and methylcobalamin for the reduction of elevated Hcy in patients presenting for chronic wound management.
Homocysteine antagonizes NO production via multiple pathways that include inhibition of arginine transport,⁴¹ pro-oxidant behavior,⁴² and by inhibition of the breakdown of the NOS inhibitor asymmetric dimethylarginine (ADMA).⁴³ Homocysteine inhibits dimethylarginine dimethylaminohydrolase (DDAH), which is responsible for degrading ADMA. The findings of the present study suggest that elevated Hcy is correlated with significant reductions in wound NO bioactivity by measurement of wound fluid NOx. Significantly decreased wound NO bioactivity could lead to impaired wound healing by inhibiting NO production, wound granulation tissue formation, VEGF-mediated wound angiogenesis, collagen deposition, epidermal migration, and re-epithelization.^1–4,16,44
Elevated serum Hcy may also inhibit wound repair by altering normal thrombosis and by occupying the fibronectin domain of fibrin during provisional wound matrix formation.⁴⁵ These studies demonstrated that Hcy binds to fibronectin by up to 62% and that Hcy binding is increased with increasing Hcy concentration.⁴⁵ These alterations significantly threaten the structural integrity of the wound matrix, as the appearance of fibronectin and the appropriate integrin receptors that bind fibronectin, fibrin, or both to fibroblasts appears to be the rate-limiting step in the formation of wound granulation tissue.⁴⁶ Homocysteine has also been observed to inhibit angiogenesis⁴⁷ and may impair collagen metabolism.⁴⁸ The combined effects of elevated Hcy on the inhibition of wound NO and, perhaps, the competitive inhibition of fibronectin-binding sites for fibrin may represent mechanisms by which elevated Hcy impairs wound healing (Figure 6).

Conclusion

The findings of this preliminary clinical study suggest that untreated, elevated Hcy should be considered an important risk factor for impaired wound healing with dermal substitute therapy and may be a common finding among chronic wound patients. A significant feature of elevated Hcy-mediated wound healing impairment may be its association with significantly decreased wound NO bioactivity, which is mediated by multiple pathways. Deficient wound NO bioactivity may be evaluated by use of wound fluid NOx measurements, which may also function as a diagnostic tool for the prediction of impaired wound healing and a method of monitoring wound treatments designed to promote the healing of chronic wounds. This study also provides evidence that the correction of elevated Hcy in a patient with impaired wound healing may promote increased wound NO bioactivity and the return of normal healing processes. Further research is needed to evaluate the relative contribution of the multifaceted aspects of elevated Hcy that antagonize NO production, wound NO bioactivity, and possibly the integrity of provisional wound matrix formation. The combination therapy of L-methylfolate, pyridoxal 5’-phosphate, and methylcobalamin is recommended as an effective treatment for lowering elevated Hcy when it is encountered in the chronic wound patient.

Acknowledgment

The authors thank Ms. Lennie J. Samsell for technical assistance in the performance of laboratory measurements for these studies and also acknowledge the assistance of the nursing staff of the HCA/Retreat Hospital Wound Healing Center for patient management and data collection performed in this study.
This study was funded in part by the National Institutes of Health (NIH), Bethesda, Md (NIH Grant R01-DK56843).