Effect Of Serum Holotranscobalamin And Methylmalonic Acid On Diagnosis Vitamin B12 Deficiency Of Diabetic Peripheral Neuropathy

BACKGROUND AND OBJECTIVEVitamin B12 deficiency can cause problem with nervous system to those found in diabetic patients.When caused by Vitamin B12 deficiency this can improve with B12 therapy.However B12 deficency is still difficult to diagnose using standard laboratory tests and the level of true deficienvy may be higher than presently thought.Severe vitamin B12 deficiency is not usually difficult to diagnose.The findings of megaloblastic anaemia and typical neurological symptoms in conjunction with a low serum B12 makes the diagnosis straightforward. However it has become increasingly apparent that these constitute only a minority of patients presenting and that there is no 'gold standard' on which to base the diagnosis of less severe cases.Studies have shown that between 25%and 38%of patients with proven B12 deficiency lacked evidence of macrocytosis and up to 44%lacked anaemia.Significant neurological symptoms can occur in the absence of anaemia and macrocytosis.Low serum B12 levels have been shown to be non-specific with between 25%and 75%of patients in some studies investigating consecutive low B12 levels showing no biochemical evidence(raised methylmalonic acid or raised homocysteine)to support a diagnosis of deficiency.There is evidence that clinically significant B12 deficiency can occur in the presence of low normal serum B12 levels. The classical causes of severe vitamin B12 deficiency include Pernicious anaemia and previous gastric or terminal ileum surgery.Less severe deficiency may be caused by food malabsorption where the B 12 is not cleaved from carrier proteins while in the stomach and is therefore not available to bind with intrinsic factor.Decreasing gastric acidity secondary to age or common drugs such as omeprazole/lansoprazole are implicatedin this process. Food malabsorption may explain the high levels of borderline B12 status in the elderly.Its clinical significance remains uncertain.Vitamin B12 deficiency may cause peripheral neuropathy and painful neuritis clinically indistinguisable from those found in diabetic neuropathy.Metformin is commonly used in the treatment of type 2 diabetes.Prolonged use may result in the malabsorption of vitamin B12.However the prevalence and clinical significance of this potential adverse drug reaction is unknown. Previous studies have shown patients on Metformin to have lower levels of serum B12 but with no difference in the most specific biochemical marker the serum Methylmalonic acid(MMA).These studies were small and may not have controlled adequately for the various confounding factors.Many centres include assessment of the serum B12 level as part of their annual diabetic review.Other centres have opted to treat all patients with diabetic neuropathy with B12 supplements.This project will be investigating the value of several laboratory tests,such as serum holotranscobalamin(holoTC)and MMA,and using these tests to see whether the rate of Vitamin B12 deficiency is higher than previously thought. If this is the case we will be looking into whether any particular group of patients are more likely to be affected. Diabetes mellitus(DM)is a common endocrine and metabolic disease,and the disorders of glycometabolism,lipometabolism and proteometabolism lies in it.Diabetic peripheral neuropathy(DPN)is one of the chronic diabetic complications.The definition of DPN is the presence of symptoms and/or signs of peripheral nerve dysfunction in people with diabetes,after exclusion of other causes.People with DM develop several types of peripheral neuropathy,including a distal sensory motor polyneuropathy,autonomic neuropathy and mononeuropathy.Estimates of its prevalence vary,it is generally accepted that clinical DPN is found in approximately 30%of people with diabetes.In the UK,the risk of foot ulceration,alate complication of neuropathy,has been estimated to be 3%per patient per year.Neuropathy also has a major impact on healthcare costs.So there is an urgent need for improvement of the diagnosis and clinical management of neuropathy in primary.As we all know,at least half of the amputations in people with diabetes are believed to be preventable,yet there are little effective method for DPN'therapy. Part 1 Influence on Metformin to Vitamin B12 Lever in Type 2 DiabetesOBJECTIVE1)Effect on metformin to vitamin B12 lever.2)Influence on dosage and duration of taking metformin to surum vitamin B12 lever.3)Influential factor of vitamin B12.METHODS1)A total of 202 eligible patients,with type 2 diabetes,in the University Hospital of Wales,Cardiff University were enrolled to the study.The diabetic patients were classified into two groups:metformin group and non-metformin group.At the same time,we recorded the duration of taking metformin and daily dose of it.2)The hemoglobin,MCV,HbAlc,lipid and renal function were detected.3)The vitamin B12,folate and ferritins were done with radio- immunoassay method on a Beckman Coulter UniCel Dx I 800 analyser.Meamwile,the serum holoTC and MMA were deteced.RESULTS1)The serum vitamin B12 concentrations were(219.11±105.36)ng/L in metformin group and(281.41±95.07)ng/L in non-metformin group.The statistical significance was revealed in the two groups(P＜0.001).2)There was no statistically significance between dosage and duration of taking metformin to surum vitamin B12 lever.3)With correlation analysis,vitamin B12 levers were correlated with BMI (P＜0.05),hemoglobin(P＜0.05),ferritin(P＜0.001),holoTC(P＜0.0001) and MMA(P＜0.0001).4)With linear regression analysis,the holoTC,ferritin,MMA and hemoglobin were the distinguished factors on serum vitamin B12 lever (R~2=0.5437).CONCLUSIONS1)Prolonged use of Metformin can result in malabsorption of vitamin B12. Therefore,vitamin B12 supplement is suggested for diabetic patients who are receiving metformin medication.2)There was no statistical significance between dosage and duration of taking metformin to surum vitamin B12 lever.3)A significant correlation is present between vitamin B12 and serum holoTC,MMA,hemoglobin and ferritin. Part 2 Apply Symptoms Questionnaires of NTSS-6A and S-LANSS in Diagnosis of Diabetic Peripheral NeuropathyOBJECTIVE1)Investigate the influential factors of diabetic peripheral neuropathy (DPN).2)The value of 10g monofilament and neurothesiometer in diagnosis of DPN.3)Evaluate the value of two symptoms questionnaires in diagnosis of DPN.METHODS1)Detecting light pressure with 10g monofilament and taking vibration test with neurothesiometer.2)According to the results of 10g monofilament and neurothesiometer,the patients were divided into two groups:DPN group and non DPN group. The influential factors were analysed by Logistic regression.3)Asking the patients to finish two forms:Neuropathy Total Symptom Score Self-Administered Version(NTSS-6A)Symptoms Questionnaires and The Leeds Assessment of Neuropathic Symptoms and Signs(S-LANSS).RESULTS1)A significant correlation is presented between DPN and duration of diabetes,age,weight,vitamin B12 lever and gender in Logistic regression analysis.2)The sensitivity for 10g monofilament and neurothesiometer is 95.29%and the specificity is 92.24%in diagnosis of DPN.The area under the curve is 0.992 and the diagnostic value is very high.3)The questionnaire NTSS-6A is better than S-LANSS.With ROC curves,the sensitivity for NTSS-6A symptoms questionnaire is 60.71%and the specificity is 66.96%in diagnosis of DPN.The area under the curve is 0.67 and the diagnostic value is low.CONCLUSIONS1)The significant influential factors of DPN were uration of diabetes,age, weight,vitamin B12 lever and gender.2)It's very useful for 10g monofilament and neurothesiometer in diagnosis of DPN.3)The two questionnaires have limited value in diagnosis of DPN. Part 3 Evaluate the Value of Serum Holotranscobalamin and Methylmalonic Acid in Diagnosis of Vitamin B12 DeficiencyOBJECTIVE1)To investigate the value of serum holotranscobalamin(holoTC)on diagnosis of vitamin B12 deficiency.2)To investigate the value of serum methylmalonic acid(MMA)on diagnosis of vitamin B12 deficiency.METHODS1)The holoTC was done with radioimmunoassay on a Abbot Axipin Analyser.2)Solid phase sample extraction for rapid determination of MMA in serum by stable isotope dilution.3)Evaluate the value of serum holoTC and MMA on diagnosis of vitamin B12 deficiency with ROC curve.RESULTS1)Defined serum holoTC＜38pmol/L as vitaminB12 deficency:The sensitivities,specificities and area under the curves(AUCs)for serum vitaminB12 and MMA levers were 72.72%,75.34%,0.792 and 58.00%, 70.00%,0.686 respectively.2)Defined serum MMA＞0.42μmol/L as vitaminB12 deficency:The sensitivities,specificities and AUCs for serum vitaminB12 and holoTC levers were 55.77%,74.50%,0.678 and 44.23%,82.52%,0.638 respectively.3)Defined serum VitaminB12＜130ng/L as vitaminB12 deficency:The sensitivities,specificities and AUCs for serum holoTC and MMA levers were 73.08%,79.88%,0.891 and 46.15%,84.05%,0.677 respectively. 4)Divided into different 4 groups based on the established cutoff (holoTC＜38pmol/l,MMA＞0.42μmol/l,VitB12＜130ng/l),observing the concentration of VitB12,MMA and holoTC in each group.Abnormality with two results together,it will be useful for the diagnosis of VitB12 deficiency.CONCLUSIONS1)Defined serum holoTC as vitaminB12 deficency,the sensitivities and specificities for serum MMA levers were low.Its value was not much better than that of vitamin B12.2)Defined serum MMA as vitaminB12 deficency,the sensitivities and specificities for serum holoTC levers were low.Our finding show the measurements of serum holoTC lever were not significantly better than a measurement of vitamin B12 concentration.3)Defined serum vitaminB12 lever as vitaminB12 deficency,the measurements of serum holoTC lever were better than a measurement of MMA concentration for predicting diabetic peripheral neuropathy.The value of holoTC is acceptable in diagnosis of vitamin B12 deficiency.4)Abnormality with two results,VitB12,MMA and holoTC,together,it will be useful for the diagnosis of VitB12 deficiency.