Serum Methylmalonic Acid and Holotranscobalamin-II as Markers for Vitamin B12 Deficiency in End-Stage Renal Disease Patients

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1 Annals of Clinical & Laboratory Science, vol. 43, no. 3, 2013 Available online at Serum Methylmalonic Acid and Holotranscobalamin-II as Markers for Vitamin B12 Deficiency in End-Stage Renal Disease Patients Navaid Iqbal 1, David Azar 1,2, Yeo-Min Yun 3, Omar Ghausi 1,2, Joachim Ix 1,2, and Robert L. Fitzgerald 1,2 1 VA Health Care System-San Diego, 2 University of California-San Diego, CA, USA; 3 Konkuk University, Seoul, Korea Abstract. Background: Vitamin replacement, particularly B vitamins, remains an important concern in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis. Serum markers such as methylmalonic acid (MMA) and holotranscobalamin (holotc) used to detect vitamin B12 deficiency are affected by impaired renal function which makes the interpretation of these biomarkers difficult in ESRD patients. We investigated the role renal failure has on MMA and holotc concentrations and evaluated using MMA and/or holotc to identify B12 deficient patients. Materials and methods: We evaluated the utility of serum MMA and holotc for its role in the detection of vitamin B12 deficiency in dialysis patients (n=17) by using the reduction of MMA concentrations as a marker of the response to vitamin B12 treatment (1 mg, intramuscular injections once per month for 3 months). Nerve conduction studies (NCS) were done before and after vitamin B12 treatments to evaluate for any alteration in peripheral sensorimotor nerve function within a subset of the cohort. Results: Receiver operating characteristic curves for detection of vitamin B12 deficiency in dialysis patients showed that serum MMA concentrations had the greatest predictive potential (area under the curve = 0.792, p = 0.043) with an optimal cutoff of 750 nmol/l. Dialysis patients (n=10) with pre-mma > 750 nmol/l and pre-holotc < 260 pmol/l showed a significant response to the vitamin B12 treatment (a mean MMA reduction of 461 nmol/l after B12 supplementation; p = 0.006). Conclusion: MMA is viable marker of B12 deficiency in ESRD patients. Holo TC has potential as a supplementary marker with MMA to predict the response of vitamin B12 supplementation. Future studies on MMA and B12 should be done to confirm these findings in larger cohorts and to identify individuals who may benefit from vitamin B12 supplementation. Key words: Methylmalonic acid, holotranscobalamin, vitamin B12, end-stage renal disease 243 Introduction Vitamin B12 is an essential cofactor involved in multiple biologic functions. B12 serves as a methyl transporter in the conversion of homocysteine to methionine by methionine synthase and in the regeneration of the active form of folic acid. B12 also serves as a cofactor to methylmalonyl-coa mutase which converts methylmalonyl-coa to succinyl- CoA, an important step in the extraction of energy from proteins and fats. Address correspondence to Muhammad N Iqbal, MD; VA Medical Center-San Diego; 3350 La Jolla Village, San Diego, CA 92161, USA; phone: x 3127; fax: ; e mail: niqbal@ ucsd.edu Sequelae of B12 deficiency include megaloblastic anemia which is secondary to ineffective erythrocyte DNA synthesis and peripheral neuropathy through ineffective nerve-sheet myelination. Unfortunately, patients remain asymptomatic until they develop a significant tissue deficiency, which may take many years. Neurologic deficits associated with vitamin B12 deficiency are well documented [1]. Deficiency leads to demyelination of spinal nerves, particularly the posterior and lateral columns, resulting in a loss of proprioceptive and vibratory sensation. In addition, there is a loss of myelin sheet within the cerebral white matter and from the peripheral nerves [1]. Prior investigations have identified reproducible neurophysiologic signs of demyelination and axonal loss due to /13/ by the Association of Clinical Scientists, Inc.

2 244 Annals of Clinical & Laboratory Science, vol. 43, no. 3, 2013 Table 1. Characteristics and the biochemical markers of the B12 status in the study population and the previously published data. This study Previously published data* Study subjects Dialysis patients Dialysis patients Healthy Vegetarians Elderly Chronic Dialysis (B12 Tx Pre) (B12 Tx Post) controls renal renal patients patients Number Age, years 63 (51-83) 63 (51-83) 34 (21-68) 46 (18-72) 82 (69-92) 66 (49-79) 68 (48-80) Females, n (%) 0 (0%) 0 (0%) 46 (49%) 62 (56%) 87 (69%) 43 (47%) N/A Vitamin B12, pmol/l ( ) ( ) ( ) ( ) ( ) ( ) ( ) HoloTC in serum, pmol/l (89-468) ( ) (17-114) (2-92) (17-320) (36-221) (50-196) MMA in serum, nmol/l ( ) ( ) (95-409) ( ) ( ) ( ) ( ) Creatinine, N/A umol/l ( ) ( ) ( ) ( ) ( ) ( ) Data are median (5 th -95 th ) percentiles except renal patients (10 th -90 th percentiles) Previously published data* B12 deficiency [2]. Furthermore, prior studies have documented both subjective improvement in neuropathy and improvements in neuromuscular testing following B12 supplementation in deficient patients [3]. It has been shown that the serum B12 concentration has low sensitivity and specificity for tissue deficient in B12 [4,5]. In contrast, serum methylmalonic acid (MMA) concentrations are increased relatively early in the course of B12 deficiency [6,7]. In otherwise healthy patients, serum MMA has been shown to be a highly sensitive and specific surrogate for tissue B12 status [8]. In patients with adequate tissue B12, MMA remains below the upper normal limit (340nmol/L). MMA is consistently elevated with tissue B12 deficiency, often in patients with serum B12 concentrations at the lower end of normal. Additionally, MMA is inversely correlated with serum B12 concentration over this range [9,10] % of serum B12 is bound to Transcobalamin-II; the B12-transcobalamin complex referred to as holotranscobalamin (holo-tc). Holo-TC is currently understood to be the active form of B12 available to tissues. As such, low holo- TC has been recognized as an early marker of vitamin B12 depletion and may show changes concurrent with or even prior to MMA elevation [11-13]. Vitamin replacement, particularly B vitamins, remains an important concern in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis. ESRD patients are particularly susceptible to vitamin deficiency due to the low B vitamin content of typical renal failure diets, the toxic and muscle wasting effect of chronic uremia, and the loss of water soluble vitamins through the dialysis procedure, which is believed to be considerably higher than that of normal urinary excretion [14]. While the concentration of MMA has been demonstrated to be affected by gender and age, increasing amounts of evidence suggests that impaired kidney function may also lead to an increase in serum MMA [15]. Large population based studies have shown a strong positive correlation between plasma levels of MMA and creatinine [12,16-20]. This is believed to be due to impaired glomerular filtration and tubular secretion and thus subsequently altered reabsorption by the renal tubules [18]. For this reason, there is continued debate over the usefulness of MMA in the setting of chronic kidney disease with specific

3 Markers for vitamin B12 deficiency in end-stage renal disease patients 245 Table 2. Mean concentrations of methylmalonic acid (MMA), holotranscobalamin (TC), and vitamin B12 (B12) pre and post vitamin B12 supplementation. P values represent paired t test results pre and post vitamin B12 supplementation. Patient# Pre MMA Post MMA Pre TC Post TC TC Pre B12 Post B12 B12 (nmol/l) MMA change (pmol/l) (pmol/l) change (pmol/l) (pmol/l) change (nmol/l) (%) (%) (%) 1 1, , , ,795 1, ,300 1, ,400 1, , , , , ,164 1, , ,155 1, , , ,000 2,000 0 Median p= < < implications for ESRD patient on chronic hemodialysis [12-13,16]. A more limited number of studies have evaluated holotc in different clinical settings. At least one report has suggested that holotc may be elevated by an unknown mechanism in ESRD patients [13]. No model has yet been developed to adjust serum MMA concentrations and holotc in patients with ESRD; thus, we do not know how to use these markers to determine the tissue B12 status of these patients. The goal of this study was to evaluate MMA and holotc as potential markers for identifying B12 deficiency in ESRD patients. Materials and Methods ESRD patients (n=17) undergoing maintenance hemodialysis (3x/week) in the hemodialysis unit at the Veterans Affairs Healthcare System in San Diego California were enrolled. MMA and holotc concentrations were measured on routinely drawn pre-dialysis serum specimens. Other pertinent serum laboratory values (e.g. B12, folate, homocysteine, iron panel, hemoglobin/hematocrit, mean corpuscular volume, creatinine, blood urea nitrogen, etc.) were also obtained from the same pre-dialysis specimen. MMA values were determined by isotope-dilution mass-spectrometry. HoloTC concentrations were determined by immunoassay (Axis-Shield Diagnostics, UK). The remaining serum chemistry values were determined by their respective FDA approved standard laboratory assays. This study was approved by the University of California-San Diego human research protection programs institutional review board. For the first three months of the study, patients received routine clinical care and monthly laboratory pre-dialysis values were recorded to establish baseline parameters. Starting at month 4 of the study, patients were dosed with standard 1 mg IM B12 injections. The intramuscular (IM) B12 injections continued for a total of 3 months and laboratory values were recorded. These timeframes were selected because previous studies have demonstrated significant changes during similar timeframes [8,21]. A minimum of 2 out of 3 presupplementation time points and 2 out of 3 postsupplement time points were required for the subject s data to be included in the data analysis. Subjects were dosed with Vitamin B12 by the nephrologist in the hemodialysis unit at the hospital. The B12 intramuscular injection was approved for clinical use in the VA San Diego Healthcare System. We chose the IM route of administration to ensure dosing compliance and limit differences in B12 absorption by oral routes of administration. Subjects (N=9) who

4 246 Annals of Clinical & Laboratory Science, vol. 43, no. 3, 2013 studies have demonstrated that the peroneal motor response recorded at EDB to be a more sensitive indicator of polyneuropathy in ESRD patients on HD, this study is designed to demonstrate the improvement in NCS parameters [23]. Thus, the tibial motor response recorded at abductor hallucis was chosen as it is more likely to be preserved in this patient population, who likely have an underlying polyneuropathy. EMG was not performed given the many confounding factors that obfuscate meaningful interpretation. Figure 1. Receiver operator characteristic (ROC) curves for ability of markers of vitamin B12 status to predict the vitamin B12 treatment response (more than 18% decrease of MMA concentration). As demonstrated in the figure, pre-mma concentrations had the greatest potential (area under the curve = 0.792, p = 0.043) with an optimal cutoff of 750 nmol/l. agreed to have nerve conduction studies (NCS) performed underwent neurophysiologic testing to evaluate peripheral nerve function under the direction of a neurologist with subspeciality training in this field. These subjects underwent NCS testing once during the three-month pre-supplementation leg of the study to establish baseline values and again after three months of B12 supplementation. While many patients may have exhibited polyneuropathy related to their respective comorbidities, comparing their neurophysiologic test results prior to and following the supplementation allowed correction for other sources of neuropathy and highlighted any potential resolution of any B12 mediated neuropathy. Previous studies have documented improvement in B12 related neuropathy following three months of supplementation [3]. Hemodialysis was performed using polysulfone dialysis membranes. Prior studies have shown that post-dialysis NCVs modestly improved after one week of HD using non-cellulosic membranes [22]; however, our patient populations are not HD naïve and they have been using the same non-cellulosic membrane for many months. Thus, the effect of pre- vs. post- hemodialysis is unlikely to be a significant confounding factor. Additionally, Mg containing dialysate has been demonstrated to decrease NCV (conversely, Mg free dialysis increased NCV) [23]. The dialysate used at VAMC San Diego is Mg containing. Electrophysiology testing was standardized for temperature, side of testing, stimulation protocol, averaging of sensory potentials, and measurement of latencies and amplitudes. Bilateral sural and radial sensory NCS were performed, in addition to bilateral tibial motor studies, and were recorded at abductor halluces. Sensory NCS were performed antidromically. Sensory nerve action potentials (SNAP) tested were bilateral sural and radial responses. Compound motor action potentials (CMAP) tested were bilateral tibial responses. Although prior Secondary end points were the summed/individual change in sensory nerve action potential (SNAP) peak latency onset from the baseline of bilateral sural and radial sensory nerves in addition to summed/individual change in the compound motor action potential (CMAP) amplitudes and distal motor latencies from the baseline of bilateral tibial motor studies. Changes in sensory nerve amplitude were not included as a secondary endpoint given the inherent wide variance of amplitude within individual SNAPS when tested serially. Results There were a total of 37 patients enrolled in the study. Of these, twenty patients dropped out of the study. The reasons varied from inability to get pre and post blood samples for B12 and holotc analysis to the inability to administer B12 injection as part of their treatment and failure of the participants to continue the study further. Four participating patients died during the study due to complications associated with renal failure. The demographic distribution of patients was based on various characteristics including age, gender, race, weight, height, heart rate, etc. Participants were all males (17 [100%]) and most were white (9 [52.9%]) and Diabetic (10 [58.8%]). As shown in Table 1, we compared the characteristics and biochemical markers of B12 status with previously published data reported by Hermann et. al.[13]. Table 2 shows the mean concentrations of serum MMA, holotc, and B12 pre and post vitamin B12 injections for each individual patient. The median MMA, holotc, and B12 values in all dialysis patients before treatment was 930nmol/L, 196pmol/L, and 543pmol/L respectively. After treatment with IM B12 injections for 3 months, the median vitamin MMA, holotc, and B12 values in dialysis patients was 750nmol/L, 312pmol/L, and 998pmol/L respectively. Following Vitamin B12 supplementation, concentrations of MMA, holotc, and B12 changed significantly (paired t-test, p=0.008, <0.001, and <0.001 respectively).

5 Markers for vitamin B12 deficiency in end-stage renal disease patients 247 Figure 1 shows that when the markers of vitamin B12 status in dialysis patients were assessed for the ability to predict the vitamin B12 treatment response (more than an 18% decrease of MMA following vitamin B12 supplementation), pre-mma (MMA concentration before the vitamin B12 treatment) had the greatest predictive potential (area under the curve = 0.792, p = 0.043) with best cutoff value of 750 nmol/l in the receiver-operating characteristic (ROC) curves. The cutoff of 18% change in the MMA level for the vitamin B12 treatment response was calculated using isotope-dilution mass-spectrometry (coefficient variation of 6%) which is three times higher the precision of the MMA assay. Three times the precision demonstrates that the measurements were analytically different at a 95% confidence interval [24] thus any values which exceed an 18% change represent a physiologic change in the concentration of MMA. Serum concentrations of pre-holotc (holotc concentration before the vitamin B12 treatment) correlated negatively to that of pre-mma (r = -0.54, p = 0.026). Using this regression line, the cutoff of holotc prior to supplementation, which corresponded to the 750 nmol/l MMA cutoff,,was determined to be 260pmol/L. Based on the cutoff concentrations of a pre-mma and a pre-holotc, out of 17 renal patients on dialysis, 10 patients with pre-mma > 750 nmol/l and pre-holotc < 260 pmol/l (59%) showed a significant MMA reduction after vitamin B12 treatment (a mean MMA reduction of 461 nmol/l; p = 0.006). The dialysis patients with biomarkers in the other ranges (initial MMA > 750 nmol/l and holotc > 260 pmol/l, MMA < 750 nmol/l and holotc < 260 pmol/l and MMA < 750 nmol/l and holotc > 260 pmol/l) did not show a significant change in MMA levels before and after vitamin B12 treatment (Figure 2). It is well documented that ESRD patients with marked B12 deficiency develop significant neurologic deficits [1-3]. In the present study we assessed changes in the neurophysiologic parameters in a subset of these patients following B12 administration. Table 3 shows that there was no statistically significant improvement in the compound motor Figure 2. Effect of intramuscular vitamin B12 injection of serum MMA (mean ± SEM) in dialysis patients. Mean MMA levels in dialysis patients with pre-mma > 750 nmol/l and pre-holo TC < 260 pmol/l (59%) (, n = 10) manifested a significant decrease (a mean MMA reduction of 461 nmol/l; paired t-test, p = 0.006). The dialysis patients with biomarkers in the other ranges [initial MMA > 750 nmol/l and holotc > 260 pmol/l (, n = 2), MMA < 750 nmol/l and holotc < 260 pmol/l ((, n = 3) and MMA < 750 nmol/l and holotc > 260 pmol/l (, n = 2)], did not show a significant change in MMA levels before and after vitamin B12 treatment (a mean MMA change of 63, 91, -102 nmol/l, respectively). action potential amplitude, distal motor latency, or sensory nerve action potential peak latency onset following B12 administration in this small patient cohort. Post-hoc power calculations were made for motor action tests and nerve function variables using the sample sizes of the subsets of subjects with these measurements. The sample sizes were sufficient to have 80% power to detect effects in the range of 1.3 to 2.1 m/s. It should be noted, however, that the nerve conduction studies were a secondary endpoint of this study which was powered to observe potential changes in MMA and holotranscobolamin following B12 supplementation.

6 248 Annals of Clinical & Laboratory Science, vol. 43, no. 3, 2013 Table 3. Comparison of pre and post nerve conduction studies in a subset of patients. P values represent paired t-test pre and post vitamin B12 supplementation. NM = Not measurable. Patient Pre Post Right Pre Post Left Pre Post Motor Pre Post Motor # Sensory Sensory Radial Sensory Sensory Radial Motor Motor Ampli Motor Motor Ampli Right Right Ampli Left Left Ampli Ampli Ampli tude Ampli Ampli tude Radial Radial tude Radial Radial tude tude tude Change tude tude Change Ampli Ampli Change Ampli Ampli Change Right Right Right Left Left Left tude (uv) tude(uv) (%) tude (uv) tude (uv) (%) Tibial Tibial Tibial Tibial Tibial Tibial Ankle Ankle Ankle Ankle Ankle Ankle (uv) (uv) (%) (uv) (uv) (%) NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM 4.75 NM NM NM NM NM NM NM NM NM 4.9 NM NM NM NM NM NM NM p = Discussion Vitamin B12 deficiency can be a major medical problem in ESRD patients. Methylmalonylcoenzyme A mutase activity is dependent on cobalamine concentrations and an elevated serum MMA level can be used as a sensitive marker of vitamin B12 deficiency [28]. Studies have reported that patients with CKD or ESRD have higher mean serum concentrations of MMA and homocysteine, despite having normal plasma concentrations of vitamin B12 or holotc [25-27]. Abnormal concentrations of MMA can be normalized in some patients with therapeutic doses of vitamin B12 supplementation, which suggests a deficiency before starting treatment [13]. A reduction of MMA by more than 270 nmol/l after vitamin B12 administration could be a proper way to detect a pretreatment deficiency in patients with renal disorders [13]. Since patients with ESRD may have higher mean MMA concentrations that are not associated with vitamin B12 deficiency, we examined the effect of B12 supplementation on concentrations of MMA in this patient population. Previous publications showed that holotc cannot be used independently as a useful marker to diagnose B12 deficiency in ESRD patients [12,13]. We measured concentrations of MMA and holotc in ESRD patients before and after B12 injections and demonstrated that holo TC alone provided no significant discriminatory power for detection of vitamin B12 deficiency in ESRD patients. When we used the cutoffs for both holotc and MMA together, no additional discriminatory power was observed compared to MMA alone in the ROC curve. However, out of 12 dialysis patients who showed a high MMA level (>750 nmol/l) before vitamin B12 administration, 10 patients with low holo TC level (<260 pmol/l) showed a significant response to vitamin B12 treatment and the remaining two patients with high holo TC level did not show a significant response to vitamin B 12 supplementation. Five patients with low MMA level (<750 nmol/l) did not show a significant response regardless of holo TC concentration either. These findings suggest that even though holo TC cannot be used as an independent marker to determine vitamin B12 status in dialysis patients, it can be used as a supplementary marker in combination with MMA to predict the response of vitamin B12 administration.

7 Markers for vitamin B12 deficiency in end-stage renal disease patients 249 Conclusion In patients with ESRD, B12 deficiency remains an important concern for the clinicians as patients remain asymptomatic for many years until significant tissue deficiency and neurologic deficits occur. MMA is a viable marker of B12 deficiency in ESRD patients, and Holo TC has potential as a supplementary marker used with MMA to predict the response to vitamin B12 treatments. Future studies on MMA and holo TC should be done to confirm these findings in larger cohorts and to extend these results to broader populations of individuals who may benefit from vitamin B12 supplementation. Acknowledgements The authors would like to acknowledge Axis Shield Diagnostics for providing partial funding for this project and would also like to thank Dr. Bill Roberts at the ARUP for analyzing specimens for holo-tc concentrations. References 1. Roos D. 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