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ORIGINAL RESEARCH
Management of Diabetic Small-Fiber Neuropathy WithCombination L-Methylfolate,Methylcobalamin, and Pyridoxal 5!-PhosphateAllen M. Jacobs, DPM,1 Dunlei Cheng, PhD2
1Allen M. Jacobs & Associates, Ltd., St. Louis, MO; 2Institute for Health Care Research and Improvement, Baylor Health Care Systems, Dallas, TX
Agents used to treat symptoms of diabetic peripheral neuropathy (DPN) are only pal-liative, not disease modifying. Although studies of monotherapy with L-methylfolate,methylcobalamin, or pyridoxal 5!-phosphate suggest that each of these bioavailable B vitamins may reverse the pathophysiology and symptoms of DPN, data on the efficacyof this combination therapy are limited. Therefore, we assessed the efficacy of an oralcombination of L-methylfolate, methylcobalamin, and pyridoxal 5!-phosphate for im-proving epidermal nerve fiber density (ENFD) in the lower extremity of patients withDPN. Eleven consecutive patients with type 2 diabetes with symptomatic DPN wereassessed for ENFD at the calf by means of skin punch biopsy and then placed on twicedaily oral-combination L-methylfolate, methylcobalamin, and pyridoxal 5!-phosphate.After approximately 6 months of treatment, patients underwent follow-up biopsy. Atthe end of their treatment, 73% of patients showed an increase in calf ENFD, and82% of patients experienced both reduced frequency and intensity of paresthesiasand/or dysesthesias. This preliminary study suggests that combination L-methylfolate,methylcobalamin, and pyridoxal 5!-phosphate increases ENFD in patients with DPN.[Rev Neurol Dis. 2011;8(1/2):xx-xx doi: 10.3909/rind0267]
The prevalence of diabetes mellitus (DM) in the United States populationwas about 7.8% in 2007.1 Up to 60% of DM patients have diabetic pe-ripheral neuropathy (DPN), a major cause of disability in the United
States. DPN is primarily due to deterioration in small nerve fibers (myelinatedA-" and unmyelinated C-fibers) that mediate pain, temperature, and autonomicfunctions.2-4 Neuropathy that is mainly or entirely associated with abnormali-ties of these fibers is termed a small-fiber neuropathy (SFN).
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The most common causes of SFNare prediabetic states (such as im-paired glucose tolerance) and frankdiabetes. Although SFN is only onetype of neuropathy affecting diabeticpatients, it causes serious paresthe-sias, painful dysesthesias, and spon-taneous pain.5 Furthermore, lack ofprotective sensation of the feet oc-curs in as many as 30% of DM pa-tients at least 40 years of age. Factorsassociated with worsening of SFNsymptoms include patient age, dura-tion of DM, and glycemic dysregula-tion.6,7 Lack of protective sensationin diabetic patients puts them athigh risk for foot ulcerations andnontraumatic amputations originat-ing from undetected injury. Approx-imately 60% of all nontraumatic am-putations performed in 2004 wereon patients with DM.1,8
Lack of consensus regarding theunderlying pathophysiology and op-timal treatment of SFN has compli-
cated its management.9,10 Currently,agents used to reduce symptoms ofpain and painful dysesthesias of SFNinclude anticonvulsants, tricyclicantidepressants, selective serotoninreuptake inhibitors, serotonin-norepinephrine reuptake inhibitors,and opioid or opioid-like analgesics.Among these medications, only du-loxetine and pregabalin have beenapproved by the US Food and DrugAdministration (FDA) for treatmentof these symptoms. Unfortunately,there is no evidence that any of theseagents modify the underlying patho-physiology of SFN. Thus, these med-ications exhibit merely palliative ac-tivity for SFN. Clearly, there is a needfor agents that modify the underly-
ing pathophysiology of DPN and,thus, SFN.11
Despite the lack of certainty aboutthe pathophysiology of SFN, oxida-tive stress and nerve perfusiondeficits appear to play importantroles in it. One model proposes thathyperglycemia-induced oxidativestress in diabetic patients causes a de-pletion of vasodilating nitric oxide(NO), which, in turn, causes “en-dothelial dysfunction” and, conse-quentially, hypoperfusion of the mi-crovasculature supplying nerves andperipheral nerve trunks. This hypo-perfusion is believed to promoteneuropathy and patient vulnerabil-ity to undetected injury, foot ulcera-tion, and resultant lower extremityamputation.11
The theory that the pathogenesisof SFN involves oxidative stress andreduced nerve perfusion led to inter-est in L-methylfolate, methylcobal-amin, and pyridoxal 5!-phosphate
(metabolically active forms of folate,B12, and B6, respectively) as potentialdisease-modifying agents in SFN.Limited evidence suggests that L-methylfolate relieves endothelial
dysfunction in patients with type 2DM by improving the activity of en-dothelial NO synthase (eNOS) and,thus, the bioavailability of NO.12
Also, L-methylfolate may eliminateproducts of oxidative stress, such asfree radicals, including superoxide
anion and peroxynitrite.13 Orallyadministered methylcobalamin ap-pears to promote myelin nodegenesis and nerve regeneration andreduce paresthesias and dysesthesiasin SFN.14,15 Pyridoxal deficiency hasbeen shown to be associated withsymptomatic SFN.16
Improvement in epidermal nervefiber density (ENFD), assessed usingthe skin punch biopsy method devel-oped by Polydefkis and colleagues,3
is a surrogate marker of improvementof DPN or, more narrowly, SFN. Thishighly sensitive and reliable methodof measuring ENFD to assess severityof SFN and response to treatment wasan ideal technique for this study. Theskin punch biopsy method, whichinvolves direct quantification ofpathologic changes in epidermalnerve fibers, is useful in identifyingSFN due to various causes, includingimpaired glucose tolerance.17-19
We hypothesized that an orallyadministered combination of L-methylfolate, methylcobalamin,and pyridoxal 5!-phosphate (LMF-MC-PP) improves ENFD and, thus,SFN, in symptomatic type 2 DM pa-tients with established SFN. The cur-rent clinical investigation was a caseseries of SFN patients in which im-munohistochemical analysis of spec-imens obtained via skin punchbiopsy was used to evaluate SFN andits response to treatment.
Patients and MethodsPatientsEleven consecutive patients withconfirmed type 2 DM, as well assymptoms consistent with SFN ofthe feet, were recruited from the pri-vate practice of Dr. Jacobs. Verbal
Currently, agents used to reduce symptoms of pain and painful dysesthesiasof SFN include anticonvulsants, tricyclic antidepressants, selective serotoninreuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, and opioidor opioid-like analgesics.
We hypothesized that an orally administered combination of L-methylfolate,methylcobalamin, and pyridoxal 5!-phosphate (LMF-MC-PP) improvesENFD and, thus, SFN, in symptomatic type 2 DM patients with establishedSFN.
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consent was obtained from eachpatient after the study, procedure,and possible complications weredescribed in detail. The study wasconducted between July 2008 andMarch 2009.
Inclusion criteria consisted of dia-betes with a history of both positiveand negative sensory symptoms (eg,paresthesias, spontaneous pain, ordysesthesias) of the lower extremi-ties. Exclusion criteria included his-tory of hereditary neuropathy, useof any medication for diabetic neu-ropathy, and presence of any type ofperipheral neuropathy that was notdue to diabetes.
Information regarding symptoms,duration of diabetes, current medica-tions, and comorbid conditions wasobtained from each patient. Each pa-tient reported symptoms consistentwith SFN, such as numbness, burn-ing, tingling, cramping, or weaknessof the lower extremities. Each pa-tient rated these symptoms at bothonset and completion of treatmentusing a visual analog scale (VAS).Also, patients underwent a full
baseline physical examination thatincluded assessment of sensitivity tovibration, light touch, and coldtemperature, as well as response to10-g monofilament.
MethodsConsecutive type 2 DM patients withsymptomatic DPN were assessedfor ENFD at the calf by means ofskin punch biopsy and then placedon twice daily oral-combination L-methylfolate (3 mg), methylcobal-amin (2 mg), and pyridoxal 5!-phos-phate (35 mg) for approximately 6months. Patients then underwentfollow-up biopsy (Table 1).
The standard biopsy protocol in-volved sterilizing the biopsy site withpovidone-iodine and anesthetizingthe site with 3 mL of injectable 0.5%bupivacaine hydrochloride withepinephrine 1:200,000. During thebiopsy procedure, two 3-mm cuta-neous punch biopsies (separated by adistance of about 5 mm) were ob-tained from a site 10 cm proximal tothe lateral malleolus between theperoneal and Achilles tendons at a
calf of each patient. Specimens werepreserved in a standard fixativesolution consisting of 2% periodatelysine paraformaldehyde and thensent for histologic analysis to Thera-path, LLC (New York, NY).
The method of ENFD evaluationrelied on immunohistochemical lo-calization of a neural antigen withinaxons. Tissue sections from the 3-mm punch biopsy specimens werecut to 50 #m in thickness andstained with polyclonal antibodiesrecognizing the protein gene product(PGP) 9.5, which is present in allnerve fibers in the skin. To obtainthe ENFD in fibers per millimeter(fibers/mm) of epidermis, patholo-gists manually counted the numberof epidermal nerve fibers in three tofive sections and divided the valueby the sum of the lengths of the epi-dermal specimens in millimeters.20
Pathologists were blinded to whetherbiopsy specimens were obtainedbefore or after treatment. The pri-mary efficacy endpoint was improve-ment in ENFD after approximately6 months of treatment.
Statistical AnalysisStatistical analysis was conducted byDunlei Cheng, PhD, using SAS® 9.2(SAS Software, Cary, NC). The re-search hypothesis was that LMF-MC-PP increases ENFD among studypatients with SFN. A Wilcoxonsigned-rank test was used to deter-mine whether, for the average pa-tient, ENFD increased significantlyafter 6 months of treatment withLMF-MC-PP. Spearman correlationbased on rank was used to determinewhether a difference in fiber densitywas related to age, sex, or duration ofdiabetes. Results were expressed inmean change in ENFD from baselineover the 6-month interval, as well asP value. In this study, a P value lessthan 0.05 was considered statisticallysignificant.
Table 1Patient Characteristics
Duration of Age of Patients Diabetes Mellitus
Patient # (y) Sex (y)
1 73 F 82 52 M 20$
3 68 M 20$
4 53 M 105 51 F 26 41 F 107 75 F % 0.58 63 F 20$
9 84 F 1010 51 F 20$
11 54 M 2
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ResultsAmong the 11 patients (4 men,7 women, aged 41-84 years), durationof type 2 DM ranged from % 6months to & 20 years (Table 1). Atbaseline, each of the 11 patients un-derwent two ENFD punch biopsiesand was then placed on one oraltablet of combination LMF-MC-PP bidfor approximately 6 months (median5.7 ' 0.6 months). Although twopost-treatment punch biopsies wereobtained from 10 of the patients, onlyone post-treatment punch biopsy wasobtained from the remaining patient(patient number 9).
Oral-combination LMF-MC-PP waswell tolerated and no patient re-ported any adverse events. Therewere no adverse events due tobiopsy.
Change in ENFD and Symptoms After6 Months of TreatmentENFD was assessed by immunohisto-chemical staining of biopsy samples.The values for average baseline, post-treatment, and increase in ENFD infibers/mm for each of the 11 patientsare shown in Table 2 and illustratedin Figure 1. The mean ENFD of the11 participants was 1.56 fibers/mmat baseline and 3.07 fibers/mm afterapproximately 6 months of oraltreatment with combination LMF-MC-PP, representing a 97% increasein ENFD (P ( .004) (Figure 2). Eight
of the 11 (73%) patients experiencedan increase in ENFD during an ap-proximately 6-month course of treat-ment with LMF-MC-PP (Figure 1).The mean per-patient increase inENFD was 1.5 fibers/mm.
Comparison of immunohisto-chemically stained sections of
epidermis revealed improvementsafter approximately 6 monthsof treatment with combination LMF-MC-PP. The photomicrographsin Figure 3 show immuno-histochemically stained baselineand post-treatment sections ofepidermis from patient 8. The post-treatment section showsregenerating small nerve fibers inboth the basement membrane andkeratin layers. Based on ENFD
measurement, the patient experi-enced a mean increase of3.75 fibers/mm during approxi-mately 6 months of treatment. Atotal of 82% of study patients re-ported reduced frequency and inten-sity of paresthesias and dysesthesiasafter 6 months of treatment.
Statistical Analysis of Changes inENFD After TreatmentFor the changes in ENFD, theWilcoxon test statistic was 22.5, witha derived P value of 0.004, which ismuch smaller than a conventionaltype I error rate of 0.05. This findingindicated that, for the average patientin the study, the increase in ENFDafter approximately 6 months of treat-ment with oral-combination LMF-MC-PP was statistically significant.The Spearman correlations were 0.30(P ( .40) between increase in ENFDand age; ) 0.36 (P ( .28) betweenincrease in ENFD and sex, favoringfemale sex (though not significantly);and 0.75 (P ( .02) between increasein ENFD and duration of diabetichistory. Thus, only duration of dia-betes correlated significantly withincrease in ENFD after therapy withLMF-MC-PP, implying that a longerduration of diabetes might be associ-ated with a greater increase in ENFDafter treatment.
Eight of the 11 (73%) patients experienced an increase in ENFD during anapproximately 6-month course of treatment with LMF-MC-PP.
Table 2Duration of Treatment, and Average Baseline, Post-Treatment,
and Increase in ENFD Values
Average Post- Average Average Treatment Increase in
aA second sample for ENFD was not obtained.ENFD, epidermal nerve fiber density.
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DiscussionPotential Benefits of LMF-MC-PP Therapy in Patients With SFNThis is the first clinical study to sug-gest that treatment with LMF-MC-PPmay promote statistically significantimprovement in ENFD in patients
with SFN. The finding that this im-provement in ENFD was associatedwith decreased anesthesia, paresthe-sia, or dysesthesia in greater than80% of study patients implies thatimprovement in these symptomswas due to increased ENFD.
These results were consistent withthose of a 2009 pilot study by Walker
and colleagues21 in which a series oftype 2 diabetic patients with SFNwho received LMF-MC-PP for 1 yearexperienced progressive and statisti-cally significant improvement incutaneous sensitivity of the feet, asmeasured by one- and two-point sta-tic Pressure-Specified Sensory Devicetesting. Together, these two studies
0
4
3
ENFD
(fib
ers/
mm
)
Patient
2
1
5
6
7
8
1 32 4 5 6 7 8 9 10 11
Baseline 6 Months Amount of Increase
Figure 1. Eight of the 11 patients (73%) experienced an increase in ENFD during an approximately 6-month course of treatment with the oral com-bination LMF-MC-PP. For patient 2, the baseline ENFD value was zero. For patients 6 and 11, both baseline and post-treatment ENFD values werezero. For patient 9, the baseline value was based on one biopsy. ENFD, epidermal nerve fiber density; LMF-MC-PP, L-methylfolate, methylcobalamin,and pyridoxal 5!-phosphate.
0
1.5
1.0
0.5
2.0
2.5
3.0
3.5
Baseline AfterTreatment
ENFD
(fib
ers/
mm
)
P ! 0.0043.07
1.56
Figure 2. The mean ENFD of the 11 patients was1.56/fibers/mm at baseline and 3.07/ fibers/mm afterapproximately 6 months of treatment with oral-combination LMF-MC-PP, representing a 97% increasein ENFD (P ( .004). ENFD, epidermal nerve fiber den-sity; LMF-MC-PP, L-methylfolate, methylcobalamin,and pyridoxal 5!-phosphate.
Baseline 6 Months
A B
Figure 3. Photomicrographs of immunohistochemically stained sections of epidermis from the left calf of patient 8,who had type 2 diabetes and diabetic small-fiber neuropathy and was treated with combination LMF-MC-PP twicedaily for 6 months. (A) Photomicrograph of baseline skin punch biopsy taken from the left calf revealed low ENFD.The basement membrane is indicated by three vertical arrows. An intraepidermal nerve fiber in the keratin layer isindicated by a horizontal triangle. (B) Photomicrograph of a similar sample from the same patient taken after ap-proximately 6 months of treatment. Based on measurement of ENFD, a mean increase of 3.75 nerve fibers/mmhad occurred during treatment. Regenerating small nerve fibers can be seen in both the basement membrane (in-dicated by four vertical arrows) and keratin layer (indicated by horizontal triangles). Original magnification x 20.ENFD, epidermal nerve fiber density; LMF-MC-PP, L-methylfolate, methylcobalamin, and pyridoxal 5!-phosphate.Image courtesy of Therapath, LLC (New York, NY). Reproduction is prohibited without written consent of Therapath.
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provide evidence that LMF-MC-PPpromotes restoration of damaged cu-taneous nerve fibers in patients withSFN.
The findings of the current studyalso are consistent with the theorythat treatment with medical nervefoods that promote the bioavailabil-ity of endothelial NO may alter theunderlying pathogenesis of DPN andconsequently SFN. As noted earlier,evidence indicates that each of thecomponents of LMF-MC-PP mayhave the potential to improveSFN by modifying its underlyingpathophysiology. Both pyridoxal 5!-phosphate and methylcobalaminmonotherapy may be essential tocritical peripheral nerve functionsthat are impaired in DPN.14,15,22
L-methylfolate has been shown toimprove endothelial function inpatients with type 2 DM, possibly bypromoting synthesis of NO.12,13,23
Pathogenesis of SFNThe pathogenesis of SFN appears tobe multifaceted and attributable tothe effects of hyperglycemia, oxida-tive stress, mitochondrial dysfunc-tion, and altered transport of nutri-ents within nerve axons as a result ofaccumulation of advanced glycationend products.24,25 Oxidative stress in-volves uncoupling the enzyme eNOSin diabetic patients and thus inter-feres with endothelial production ofNO, a gaseous free radical that medi-ates relaxation of vascular smoothmuscle and promotes tissue repair.26
As is depicted in Figure 4, reductionin NO results in constriction ofblood vessels, reduced capillary flow,nerve hypoxia, and, over time,SFN.12,13,22,27 Interestingly, reductionin NO synthesis has also been shownto be associated with impairedwound healing in diabetic pa-tients.11,27,28 Histologic findings inSFN include reduced cutaneous pe-ripheral nerve fiber branch density
and length, dermal and epidermalaxon swelling, subepidermal nerveplexus thinning, nerve terminalsprouting and encapsulation, andpresence of mural reactive basalcells.29-31
Another clue to the pathogenesisof SFN comes from studies showingthat elevated homocysteine, which is
often present in diabetic patientswith neuropathy, may be a risk factorfor DPN in general, including auto-nomic neuropathy.7,32,33 One widelyheld theory is that hyperhomocys-teinemia is directly toxic to the vas-cular endothelium and induces anendothelial dysfunction that pro-motes endothelial thrombosis and,consequently, reduced perfusion ofperipheral nerves.34 Another theory
is that hyperhomocysteinemia indi-rectly promotes SFN by disruptingendothelial synthesis of NO, asmooth muscle relaxant that facili-tates blood flow to peripheralnerves.35
Evidence that L-methylfolate,methylcobalamin, and pyridoxine5!phosphate individually reduce ho-
mocysteine and promote NO synthe-sis suggests that these B vitaminshave the capacity to alleviate bothpositive and negative symptoms ofdiabetic neuropathy. L-methylfolate,the metabolically active form of folicacid, is a cofactor for homocysteinemetabolism in that it provides amethyl group to homocysteine forreconversion to methionine36; more-over, L-methylfolate directly promotes
Figure 4. Nitric oxide synthesis and potential mechanisms of action of L-methylfolate. Diagram illustrates nitricoxide (NO) synthesis from L-arginine, as catalyzed by endothelial NO synthase (eNOS), and potential mechanismsfor beneficial effects of L-methylfolate on eNOS. The beneficial effects of L-methylfolate may be explained bydifferent mechanisms. I. Tetrahydrobiopterin (BH4) rescue or BH4 stabilization, in which L-methylfolate may stimu-late endogenous BH4 regeneration from quinoid-dihydrobiopterin (q-BH2) or lead to chemical stabilization of BH4. II. Antioxidant effects, in which L-methylfolate may act as a direct antioxidant. III. Direct effect on eNOS, inwhich L-methylfolate reduces superoxide generation and increases NO synthesis in a BH4-dependent manner.Adapted with permission from Verhaar MC, Stroes E, Rabelink TJ. Folates and cardiovascular disease. ArteriosclerThromb Vasc Biol. 2002;22:6-11.13
L-MethylfolateI.
II.
III.Homocysteine Methionine
CoupledeNOS
UncoupledeNOS
Nitric Oxide
Arginine
7,8-BH2/q-BH2
BH4
O2!
O2
Evidence that L-methylfolate, methylcobalamin, and pyridoxine 5!phosphateindividually reduce homocysteine and promote NO synthesis suggests thatthese B vitamins have the capacity to alleviate both positive and negativesymptoms of diabetic neuropathy.
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increased endothelial production ofNO.12,13
L-methylfolate in oral-combinationLMF-MC-PP offers several advan-tages over folic acid as a source offolate. First, the biologic activity of L-methylfolate is seven times thatof folic acid. Second, although folicacid must undergo a complex four-step conversion process to becomeactivated, L-methylfolate already ismetabolically active. Finally, meta-bolically active L-methylfolate bene-fits those patients who carry a ge-netic polymorphism that preventstheir converting folic acid to L-methylfolate.34,37
The formulation of LMF-MC-PPused in this study (Metanx®; Pamlab,Covington, LA) is classified by theFDA as a prescription medicalfood. The FDA defines a medicalfood as a substance that is prescribedby a physician “for the specificdietary management of a disease orcondition for which distinctivenutritional requirements, based onrecognized scientific principles, areestablished by medical evaluation.”38
LMF-MC-PP use is indicated for thedistinct nutritional requirements ofdiabetic patients with endothelial
dysfunction12,39,40 who present withloss of protective sensation andneuropathic pain41,42 associated withdiabetic peripheral neuropathy.43
Reliable Method for Diagnosing SFNCurrently, the most reliable ap-proach to diagnosing SFN involvesdemonstrating either reduction innumber or altered morphology ofepidermal nerve fibers by means ofskin punch biopsy.44-48 Direct evalua-tion of ENFD in skin obtained byskin punch biopsy is a more sensitivemethod for diagnosing SFN than useof sensory nerve conduction studiesalone.6,47,49,50 Although the sensitivi-ties of clinical examination andquantitative sensory thermal thresh-old testing for diagnosing SFN areonly 54% and 49%, respectively, thesensitivity of skin punch biopsy indiagnosing SFN is up to 88.4%. Thespecificity of skin punch biopsy indiagnosing SFN is as high as 97%.51,52
Determination of ENFD by skinpunch biopsy has a broad range ofapplications, including differentia-tion among distal polyneuropathy,sensory radiculopathy, and focalneuropathy. The technique is usefulin detecting other neuropathies,
such as those related to vasculitis,amyloid, or sarcoid, as well as moni-toring progression of neuropathy orresponse to treatment.51 Combiningquantitative sensory testing withENFD determination by skin punchbiopsy is effective in diagnosingsuspected SFN in diabetic or glucose-intolerant patients with neuropathicpain, even prior to the develop-ment of signs of either autonomicsmall-fiber or sensory large-fiberpathology.4
Assessing Theoretical Limitations andAdvantages of StudyThe current study was limited by itssmall size, method of participant se-lection, possible volunteer bias, lackof a placebo-treated group for com-parison, lack of blinding, and thesubjective nature of the VAS assess-ment. Also, the study did not controlfor differences in duration of type 2DM among participants and the pos-sibility that only some patients usedinsulin and/or a symptom-reducingmedication. Despite these limita-tions, the finding of statistically sig-nificant changes in ENFD based on ahighly valid, objective method oftesting is compelling.
Main Points• As many as 60% of patients with diabetes mellitus have small-fiber peripheral neuropathy (SFN), a leading cause of
disability in the form of paresthesias, painful dysesthesias, and lack of protective sensation, which puts these patientsat high risk for foot ulcerations and nontraumatic amputations originating from undetected injury.
• There is no evidence that agents currently used to reduce paresthesias and dysesthesias of SFN modify the underlyingpathophysiology of SFN; these medications appear to have only palliative activity in SFN.
• Studies of monotherapy with L-methylfolate, methylcobalamin, or pyridoxal 5!-phosphate suggest that these B vita-mins may reverse both the pathophysiology and symptoms of diabetic SFN.
• We hypothesized that oral combination L-methylfolate, 3 mg, methylcobalamin, 2 mg, and pyridoxal 5’-phosphate,35 mg (LMF-MC-PP), improves epidermal nerve fiber density (as determined by immunohistochemical analysis of skinpunch biopsy), in symptomatic patients with type 2 diabetes and established SFN.
• Our study found that treatment with LMF-MC-PP was associated with a statistically significant increase in epidermalnerve fiber density and a reduction in frequency and intensity of paresthesias and dysesthesias.
• The data from this pilot study involving a combination of metabolically active LMF-MC-PP are consistent with stud-ies of monotherapy with these B vitamins and suggests that LMF-MC-PP modifies the pathophysiology of diabetic SFNand improves perfusion and thus sensory perception of peripheral nerves in patients with diabetic SFN.
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ConclusionsThis study suggests that oral admin-istration of LMF-MC-PP promotes in-crease in ENFD in participants withdiabetic SFN and improves symp-toms of anesthesia, paresthesia, ordysesthesia. A high-quality, double-blinded, randomized, controlled trialis necessary to confirm the effective-ness of LMF-MC-PP in SFN. Mean-while, LMF-MC-PP should be consid-ered in the treatment of patientswith diabetic SFN in whom othertherapies have been ineffective.
The authors thank Pamlab, LLC, forproviding the samples of LMF-MC-PP usedin the clinical study. Dr. Jacobs is a memberof the speakers’ bureau for Pamlab, LLC. Dr. Cheng has no corporate financialarrangements to disclose.
References1. National Diabetes Information Clearinghouse.
National Diabetes Statistics, 2007. National In-stitute of Diabetes and Digestive and KidneyDiseases. http://diabetes.niddk.nih.gov/dm/pubs/statistics/#complications. Accessed February4, 2011.
2. Kramer HH, Rolke R, Bickel A, Birklein F. Ther-mal thresholds predict painfulness of diabeticneuropathies. Diabetes Care. 2004;27:2386-2391.
3. Polydefkis M, Hauer P, Sheth S, et al. The timecourse of epidermal nerve fibre regeneration:studies in normal controls and in people withdiabetes, with and without neuropathy. Brain.2004;127:1606-1615.
4. Vlcková-Moravcová E, Bednarik J, Dusek L, et al.Diagnostic validity of epidermal nerve fiberdensities in painful sensory neuropathies. Mus-cle Nerve. 2008;37:50-61.
5. Lacomis D. Small-fiber neuropathy. MuscleNerve. 2002;26:173-188.
6. Shun CT, Chang YC, Wu HP, et al. Skin dener-vation in type 2 diabetes: correlations with dia-betic duration and functional impairments.Brain. 2004;127:1593-1605.
7. Boulton AJ, Vinik AI, Arezzo JC, et al. Diabeticneuropathies. A statement by the AmericanDiabetes Association. Diabetes Care. 2005;28:956-962.
8. Frykberg RG, Lavery LA, Pham H, et al. Role ofneuropathy and high foot pressures in diabeticfoot ulceration. Diabetes Care. 1998;21:1714-1719.
9. Argoff CE, Backonja MM, Belgrade MJ, et al.Consensus guidelines: treatment planning andoptions. Diabetic peripheral neuropathic pain.Mayo Clin Proc. 2006;81(4 Suppl):S12-S25.
11. Veves A, Akbari CM, Primavera J, et al.Endothelial dysfunction and the expression ofendothelial nitric oxide synthase in diabeticneuropathy, vascular disease, and foot ulcera-tion. Diabetes. 1998;47:457-463.
12. van Etten RW, de Koning EJ, Verhaar MC, et al.Impaired NO-dependent vasodilation in pa-tients with Type II (non-insulin-dependent)diabetes mellitus is restored by an acuteadministration of folate. Diabetologia. 2002;45:1004-1010.
13. Verhaar MC, Stroes E, Rabelink TJ. Folates andcardiovascular disease. Arterioscler Thromb VascBiol. 2002;22:6-11.
14. Yaqub BA, Siddique A, Sulimani R. Effects ofmethyl-cobalamine on diabetic neuropathy.Clin Neurol Neurosurg. 1992;94:105-122.
15. Watanabe T, Kaji R, Oka N, et al. Ultra-highdose methylcobalamin promotes nerve regen-eration in experimental acrylamide neuropa-thy. J Neurol Sci. 1994;122:140-143.
16. McCann VJ, Davis RE. Serum pyridoxal con-centration in patients with diabetic neuropa-thy. Aust N Z J Med. 1978;8:259-261.
17. Quattrini C, Tavakoli M, Jeziorska M, et al.Surrogate markers of small fiber damage inhuman diabetic neuropathy. Diabetes. 2007;56:2148-2154.
18. Holland NR, Crawford TO, Hauer P, et al. Small-fiber sensory neuropathies: clinical course andneuropathology of idiopathic cases. Ann Neurol.1998;44:47-59.
19. Sumner CJ, Sheth S, Griffin JW, et al. Thespectrum of neuropathy in diabetes and im-paired glucose tolerance. Neurology. 2003;60:108-111.
20. Lauria G, Cornblath DR, Johansson O. EFNSguidelines on the use of skin biopsy in the di-agnosis of peripheral neuropathy. Eur J Neurol.2005;12:747-758.
21. Walker MJ Jr, Morris LM, Cheng D. Improve-ment of cutaneous sensitivity in diabeticperipheral neuropathy with combination L-methylfolate, methylcobalamin, and pyri-doxal 5!-phosphate. Rev Neurol Dis. 2010;7:132-139.
22. Sheehan P. An overview of peripheral neu-ropathy in diabetes. Vasc Dis Manag. 2009;6:68-71.
23. Mangoni AA, Sherwood RA, Asonganyi B, et al.Short-term folic acid supplementation en-hances endothelial function in patients withtype 2 diabetes. Am J Hypertens. 2005;18:220-226.
24. Brownlee M. Biochemistry and molecular cellbiology of diabetic complications. Nature.2001;414:813-820.
25. King RH. The role of glycation in the patho-genesis of diabetic polyneuropathy. Mol Pathol.2001;54:400-408.
26. Schwentker A, Billiar TR. Nitric oxide andwound repair. Surg Clin North Am. 2003;83:521-530.
27. Chen RJ, Zheng YL, Xu LS. Clinical trials on ef-fects of methylcobalamin in the treatment ofdiabetic neuropathy. Chinese J Clin Rehabil.2002;6:1280-1281.
28. Boykin JV Jr, Baylis C. Homocysteine–a stealth mediator of impaired wound healing: apreliminary study. Wound. 2006;18:1-12.
29. De Sousa EA, Hays AP, Chin RL, et al. Charac-teristics of patients with sensory neuropathydiagnosed with abnormal small nerve fibres onskin biopsy. J Neurol Neurosurg Psychiatry.2006;77:983-985.
30. Wendelschafer-Crabb G, Kennedy WR, Walk D.Morphological features of nerves in skin biop-sies. J Neurol Sci. 2006;242:15-21.
31. Ambrosch A, Dierkes J, Lobmann R, et al. Rela-tion between homocysteinaemia and diabeticneuropathy in patients with type 2 diabetesmellitus. Diabet Med. 2001;18:185-192.
32. Bruce SG, Young TK. Prevalence and risk factorsfor neuropathy in a Canadian First Nationcommunity. Diabetes Care. 2008;31:1837-1841.
33. Cohen JA, Jeffers BW, Stabler S, et al. Increasinghomocysteine levels and diabetic autonomicneuropathy. Auton Neurosci. 2001;87:268-273.
34. Venn BJ, Green TJ, Moser R, Mann JI. Compar-ison of the effect of low-dose supplementationwith L-5-methyltetrahydrofolate or folic acidon plasma homocysteine: a randomizedplacebo-controlled study. Am J Clin Nutr.2003;77:658-662.
35. Welch GN, Loscalzo J. Homocysteine andatherothrombosis patients. N Engl J Med.1998;338:1042-1050.
36. van Guldener C. Why is homocysteine elevatedin renal failure and what can be expected fromhomocysteine-lowering? Nephrol Dial Trans-plant. 2006;21:1161-1166.
37. Willems FF, Boers GH, Blom HJ, et al. Pharma-cokinetic study on the utilisation of 5-methyl-tetrahydrofolate and folic acid in patientswith coronary artery disease. Br J Pharmacol.2004;141:825-830.
38. Food and Drug Administration (FDA). Fre-quently Asked Questions about Medical Foods.U.S. Department of Health and HumanServices, Food and Drug Administration,Center for Food Safety and Applied Nutrition,May 2007. http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/MedicalFoods/ucm054048.htm.Accessed February 4, 2011.
39. Verhaar MC, Wever RM, Kastelein JJ, et al. 5-methyltetrahydrofolate, the active form offolic acid, restores endothelial function in fa-milial hypercholesterolemia. Circulation. 1998;97:237-241.
40. Romerio SC, Linder L, Nyfeler J, et al. Acutehyperhomocysteinemia decreases NO bioavail-ability in healthy adults. Atherosclerosis. 2004;176:337-344.
41. Li G. [Effect of methylcobalamin on diabeticneuropathies. Beijing Methycobal Clinical TrialCollaborative Group.] Zhonghua Nei Ke Za Zhi.1999;38:14-17.
44. Beiswenger KK, Calcutt NA, Mizisin AP. Epider-mal nerve fiber quantification in the assess-ment of diabetic neuropathy. Acta Histochem.2008;110:351-362.
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45. McCarthy BG, Hsieh ST, Stocks A, et al. Cuta-neous innervation in sensory neuropathies:evaluation by skin biopsy. Neurol (Tokyo).1995;45:1848-1855.
46. Wang L, Hilliges M, Jernberg T, et al. Proteingene product 9.5-immunoreactive nerve fibresand cells in human skin. Cell Tissue Res.1990;261:25-33.
47. Periquet MI, Novak V, Collins MP, et al. Painfulneuropathy: prospective evaluation using skinbiopsy. Neurology. 1999;53:1641-1647.
48. Ebenezer GJ, Hauer P, Gibbons C, et al. Assess-ment of the epidermal nerve fibers: a new diag-nostic and predictive tool for peripheral neu-ropathies. J Neuropathol Exp Neurol.2007;66:1059-1073.
49. Holland NR, Stocks A, Hauer P, et al. Intraepi-dermal nerve fiber density in patients withpainful sensory neuropathy. Neurology.1997;48:708-711.
50. Hermann DN, Griffin JW, Hauer P, et al. Epi-dermal nerve fiber density and sural nerve
morphometry in peripheral neuropathies.Neurology. 1999;53:1934-1940.
51. Lauria G, Devigili G. Skin biopsy as a diagnos-tic tool in peripheral neuropathy. Nature ClinPract. 2007;3:546-557.
52. Devigili G, Tugnoli V, Penza P, et al. The diag-nostic criteria for small fibre neuropathy: fromsymptoms to neuropathology. Brain. 2008;131:1912-1925.
