Increased Plasma Methylmalonic Acid Level Does Not Predict Clinical Manifestations of Vitamin B12 Deficiency

ORIGINAL INVESTIGATION Increased Plasma Methylmalonic Acid Level Does Not Predict Clinical Manifestations of Vitamin B12 Deficiency

Anne-Mette Hvas, MD; Jørgen Ellegaard, MD; Ebba Nexø, MD

Background: The prevalence of vitamin B12 defi- pants, P-MMA levels increased substantially, whereas 44% ciency, defined as an elevated concentration of plasma showed a decrease. Level of P-MMA was significantly but methylmalonic acid (P-MMA), has been estimated to be not strongly associated with levels of plasma cobala- 15% to 44% in the elderly. However, we do not know mins (r=−0.22, PϽ.001) and plasma total homocyste- whether an increased P-MMA level actually indicates or ine (r=0.37, PϽ.001). After adjustment for age and sex, predicts a clinical condition in need of treatment. we found no associations between P-MMA concentration and the total symptom score (P=.61), the total Neu- Participants and Methods: In a follow-up study, 432 rological Disability Score (P=.64), or other clinical mani- individuals not treated with vitamin B12 were examined festations related to vitamin B12 deficiency. 1.0 to 3.9 years after initial observation of an increased P-MMA concentration (Ͼ0.28 µmol/L). The examina- Conclusions: An increased level of P-MMA did not tion included laboratory tests, a structured interview to predict a further increase with time and clinical mani- disclose symptoms, a food frequency questionnaire, and festations related to vitamin B12 deficiency. We there- a clinical examination including a Neurological Disabil- fore challenge the use of an increased P-MMA concen- ity Score. tration as the only marker for diagnosis of vitamin B12 deficiency. Results: Variation in P-MMA levels over time was high (coefficient of variation, 34%). In only 16% of partici- Arch Intern Med. 2001;161:1534-1541

HE ORIGINAL concept of per- Prevalence estimates of vitamin B12 nicious anemia, defined as deficiency, defined as an elevated P-MMA lack of intrinsic factor, rep- concentration, vary widely. Studies from resents only one possible the United States suggest a prevalence of and rather rare presenta- 15% to 20% among elderly outpatients 12,13 tionT of vitamin B12 deficiency. Strong in- (P-MMA level Ͼ0.37 µmol/L), whereas centives exist to establish accurate diag- European studies suggest a prevalence of nostic tests because of the often diffuse and 39% to 44% among healthy elderly indi- 14,15 nonspecific symptoms of mild vitamin B12 viduals (P-MMA level Ͼ0.24 µmol/L) deficiency. Anemia might be absent1,2 and and a prevalence of 24% among free- damage to the nervous system might be living elderly Dutch persons (P-MMA level reversible when treated in time3 but irre- Ͼ0.32 µmol/L).16 However, it is now un- versible after delayed diagnosis.4,5 certain to which extent an increase in the Use of the deoxyuridine suppression P-MMA level actually indicates or pre- test has permitted recognition of early and dicts a clinical condition in need of treat- 17,18 mild vitamin B12 deficiency characterized ment, and we still lack consensus about by biochemical dysfunction but lack of clear a gold standard for the diagnosis of vita- 6,7 clinical features of deficiency. However, min B12 deficiency. the test has limited clinical applicability be- In the present study we questioned cause it is cumbersome to perform. Dur- the clinical significance of an increased From the Departments of ing the past 10 years, determination of P-MMA level. The study aims were to Hematology, Aarhus plasma methylmalonic acid (P-MMA) and estimate the long-term trend of an ini- Amtssygehus (Drs Hvas and Ellegaard), and Clinical plasma total homocysteine (P-tHcy) lev- tially elevated P-MMA level in individu- Biochemistry, Aarhus els has been increasingly used. Level of als who did not receive cyanocobalamin Kommunehospital (Dr Nexø), P-MMA has been suggested as a more spe- therapy and to examine the associations Aarhus University Hospital, cific and sensitive marker than levels of between clinical manifestations related 8-11 Aarhus, Denmark. plasma cobalamins. to vitamin B12 deficiency and elevated

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 PARTICIPANTS AND METHODS hemoglobin levels were 7.40 to 9.60 mmol/L for women and 8.40 to 10.80 mmol/L for men and for erythrocyte mean STUDY POPULATION cell volume was 85 to 100 fL. Plasma creatinine level was measured using the Jaffe method and a Roche Cobas Inte- From the laboratory information system (Department of gra 700 autoanalyzer (HiCo Creatinine Jaffe method; Boeh- Clinical Biochemistry, Skejby Sygehus, Aarhus University ringer Mannheim GmbH, Mannheim, Germany) (analyti- Hospital, Aarhus, Denmark) we obtained information on cal imprecision Ͻ3%); the reference intervals were 44 to 1754 individuals aged 18 years and older living in the 115 µmol/L (0.5-1.3 mg/dL) for women and 62 to 133 Aarhus municipality (283000 inhabitants) who had a µmol/L (0.7-1.5 mg/dL) for men. P-MMA level greater than the reference interval (Ͼ0.28 µmol/L) between January 1, 1995, and December 31, INTERVIEW AND CLINICAL EXAMINATIONS 1997 (prestudy P-MMA) (Figure 1). Measurement of P-MMA concentration was requested by the physician in A history of present and previous diseases was obtained. charge of the patient because of suspected vitamin B12 Information on symptoms was obtained by structured deficiency. interview. We recorded anemia symptoms (daily fatigue, To exclude individuals who had received cyanoco- palpitations, shortness of breath, and angina on effort), balamin treatment we used a 3-step procedure. From Na- gastrointestinal symptoms (reduced sense of taste, sore tional Health Insurance, Aarhus County, we received in- mouth or tongue, daily reduced appetite, daily nausea, formation on all prescribed cyanocobalamin preparations.19 and daily diarrhea), and neurological symptoms using a For all individuals not excluded by this procedure we asked slightly modified version of the Neurological Symptom their general practitioner about cyanocobalamin prescrip- Score.22 Anemia, gastrointestinal, and Neurological tions. Finally, the initial interview included questions about Symptom Scores were summed to a total symptom score. previous and current treatment with cyanocobalamin. In addition, we recorded current drug use and consump- A total of 571 individuals (33%) had received cyano- tion of alcohol. Dietary vitamin B12 intake was estimated cobalamin treatment and were excluded, and another 28 using part of a validated food frequency question- individuals were excluded because they had participated naire.23-25 in a preceding pilot study. The neurological examination comprised testing for Of 1155 individuals with no report of cyanocobalamin vibration sense, joint position sense, cutaneous sensation, treatment, we included all 336 with prestudy P-MMA levels hyporeflexia, and muscular strength. Vibration sense was of 0.40 µmol/L or greater and took a geographical sample of tested at the medial malleolus, compared with a stimulus 647 individuals from 819 with prestudy P-MMA levels of 0.29 at the processus styloideus ulnae. Joint position sense was to 0.39 µmol/L. tested at the hallux and the index finger. Cutaneous sen- Of the 983 individuals addressed, 49 reported that they sation was tested by pinprick on the pulp of the hallux and had received cyanocobalamin treatment, 21 had died, and the index finger and by light touching of the dorsum of the 1 had emigrated, leaving 912 individuals eligible for fol- foot, the shin, and the forearm. A test for the Romberg sign low-up examination. Of these, 461 individuals (51%) was performed and gait was assessed. “Finger-nose” and volunteered to participate, but 10 did not attend the fol- “heel-knee-shin” tests were performed, as was testing for low-up examination and 19 reported during the interview dysdiadochokinesis. that they had received cyanocobalamin treatment. The fol- We used a slightly modified version of the Neurologi- low-up examinations of the 432 participants were per- cal Disability Score (a summed score of muscle strength, formed between October 7, 1998, and May 31, 1999, 1.0 reflexes, and sensory loss) to quantify the degree of pe- to 3.9 years after the prestudy P-MMA measurement. ripheral neuropathy.22 The Neurological Disability Score The study was approved by the Research Ethics Com- was the sum of 28 item scores, each ranging from 0 (nor- mittee of Aarhus County. Written informed consent was mal) to 4 (high degree of impairment). obtained from all participants. In addition, the examination included assessment of the nutritional state, inspection of the oral cavity, heart and LABORATORY TESTS lung auscultation, blood pressure measurement, and ab- dominal palpation. Levels of P-MMA were measured using stable isotope– All participants were examined by the same investi- dilution capillary gas chromatography–mass spectrometry gator (A.-M.H.), who did not know the laboratory test re- (analytical imprecision Ͻ8%)20; the reference interval was 0.08 sults when the examinations were performed. to 0.28 µmol/L.21 Levels of P-tHcy were measured using an immunologi- STATISTICAL ANALYSIS cal method and Imx (Abbott Laboratories, Abbott Park, Ill) equipment (analytical imprecision Ͻ5%). Plasma was sepa- For analyses of associations among laboratory test results rated from the blood cells within 2 hours. The reference in- we used the t test (independent samples), the ␹2 test for terval was 5.8 to 11.9 µmol/L. Levels of plasma cobalamins trend, linear regression, the Pearson correlation, and the were determined using an automated chemiluminescence Levene test. To analyze the associations between the bio- system (ACS: Centaur Automated Chemiluminescence chemical markers and the clinical manifestations we used System; Chiron Diagnostics Corporation, East Walpole, Mass) linear and logistic regression. Log transformations were used and a competitive protein binding assay (analytical impre- when appropriate. Differences were regarded as statisti- cision Ͻ10%); the reference interval was 200 to 600 pmol/L. cally significant at PϽ.05. Data were entered and analyzed Standard methods were used for determination of using statistical analysis software (SPSS for Windows; SPSS hematologic parameters. Reference intervals for blood Inc, Chicago, Ill).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 P-MMA levels. (In Denmark, vitamin B12 treatment 1754 Individuals With P-MMA >0.28 µmol/L implies cyanocobalamin or hydroxocobalamin. The ≥ (Aged 18 y) 571 Had Started term cyanocobalamin used herein covers both possi- Cyanocobalamin Treatment bilities.) 28 Had Participated in 1155 a Preceding Pilot Study RESULTS Geographical Sample

Addressed: 983 PARTICIPANTS 21 Died, 1 Emigrated 49 Reported Cyanocobalamin In the 432 participants, the median prestudy P-MMA level Treatment Eligible for Follow-up Study: was 0.33 µmol/L (range, 0.29-3.60 µmol/L) and the me- 912 dian age was 72 years (range, 23-102 years). Study participation was refused by 363 individuals (median prestudy P-MMA level, 0.36 µmol/L; median age, 80 years), No Response: Refused Participation: Agreed to Participate: 88 363 461 and 88 individuals did not respond (median prestudy P-MMA level, 0.35 µmol/L; median age, 71 years). Re- 10 Did Not Attend the fusers were older (PϽ.001) and had a higher prestudy Follow-up Examination 19 Reported Cyanocobalamin Participated: P-MMA level (P=.007, prestudy P-MMA level log trans- Treatment 432 formed, t test). Four hundred three participants underwent clini- Figure 1. Study population: individuals with an increased plasma cal examination and laboratory testing and 29 under- methylmalonic acid (P-MMA) concentration (Ͼ0.28 µmol/L) between went laboratory testing only. January 1, 1995, and December 31, 1997. The study population was divided into 2 sub- groups: one group (n=118) used vitamin supplements 5.00 containing 1 to 2 µg of cyanocobalamin and the other group (n=285) took no vitamins. Using linear regression adjusted for age and sex, we found no difference between the 2 groups concerning prestudy P-MMA levels 2.00 (PϾ.99), P-MMA levels at follow-up (P=.27), or change in P-MMA levels (P=.25). All analyses between biochemi- 1.00 cal markers and clinical manifestations were performed mol/L µ for users and nonusers of supplements. No results dif- fered between the 2 groups, and we therefore present 0.50 pooled results for the whole study population.

CHANGES IN P-MMA CONCENTRATION Follow-up P-MMA, 0.20 AFTER 1.0 TO 3.9 YEARS The interval from the prestudy measurement of P-MMA 0.10 to follow-up was 1.0 to 1.9 years for 59% of participants, 2.0 to 2.9 years for 24%, and 3.0 to 3.9 years for 17%. Figure 2 shows the association between prestudy 0.05 0.10 0.20 0.50 1.00 2.00 5.00 and follow-up P-MMA levels. The correlation between µ Prestudy P-MMA, mol/L the log-transformed measurements was significant Figure 2. Association between prestudy and follow-up plasma (PϽ.001), but the variation was substantial (coefficient methylmalonic acid (P-MMA) levels in 432 individuals with a prestudy of variation, 34%, estimated from the SD of the log- P-MMA level greater than 0.28 µmol/L who were not treated with transformed ratio, follow-up vs prestudy P-MMA levels). cyanocobalamin before measurement of P-MMA levels 1.0 to 3.9 years later. The coefficient of determination (r 2) was 0.24, indicat- ing that only 24% of the variation in follow-up P-MMA levels could be explained by the variation in prestudy Table 1. Association Between Prestudy P-MMA Levels and the Change in P-MMA Levels During Follow-up* P-MMA levels. Table 1 shows the association between prestudy Change in P-MMA Level, P-MMA levels and the change in P-MMA levels during No. (%) follow-up. In general, P-MMA levels did not increase: only 16% of participants had an increase of more than 20%, Prestudy P-MMA, Ͼ20% Յ20% Ͼ20% whereas 45% had a decrease of more than 20%. Only 13 µmol/L Decrease Change Increase Total† participants (3%) had a P-MMA concentration of 1.00 0.29-0.39 138 (45) 128 (41) 44 (14) 310 (100) µmol/L or greater at follow-up. The duration of fol- Ն0.40 54 (44) 41 (34) 27 (22) 122 (100) Total 192 (44) 169 (39) 71 (16) 432 (100) low-up did not affect the magnitude of change (P=.72, ␹2 for trend). *P-MMA indicates plasma methylmalonic acid. The variation in the ratio of follow-up vs prestudy †Not all percentages equaled exactly 100. P-MMA levels was significantly higher in participants with

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 2. Distribution of Test Values and Age as Percentiles in 432 Study Participants*

Percentile

Variable Minimum 5 25 50 75 95 Maximum Prestudy P-MMA, µmol/L 0.29 0.29 0.31 0.33 0.41 0.76 3.60 Follow-up P-MMA, µmol/L 0.06 0.14 0.23 0.30 0.41 0.75 5.3 Plasma cobalamins, pmol/L 78 147 211 272 354 538 1549 P-tHcy, µmol/L 3.43 6.27 8.95 11.36 15.31 24.21 68.76 Plasma creatinine, µmol/L† 50 66 80 90 105 153 584 Blood hemoglobin, mmol/L 5.70 7.30 8.10 8.70 9.20 10.20 11.60 Erythrocyte mean cell volume, fL 72 84 89 92 95 102 118 Age, y 23.9 34.1 56.5 72.2 80.2 87.6 102.6

*P-MMA indicates plasma methylmalonic acid; P-tHcy, plasma total homocysteine. †To convert plasma creatinine from micromoles per liter to milligrams per deciliter, divide micromoles per liter by 88.4.

prestudy P-MMA levels of 0.40 µmol/L or greater than in CLINICAL MANIFESTATIONS participants with lower prestudy P-MMA levels (PϽ.001, log-transformed data, Levene test). Of 403 participants who underwent clinical examina- Plasma creatinine concentration was known at the tion, 397 (99%) were of Danish origin, 249 (62%) were time of the prestudy P-MMA measurement for 110 par- women, and 13 (3%) were living in institutions. Twenty- ticipants. No correlation was found between change in five participants (6%) had diabetes, 15 (4%) had hypo- P-MMA level and change in plasma creatinine level thyroidism, and 134 (33%) recorded cardiovascular (r=0.12; P=.21, log-transformed data). disease. The clinical manifestations were evaluated as LABORATORY TESTS: FOLLOW-UP STUDY symptoms (complaints reported by the participants) (1998-1999) and signs (manifestations recorded by the examining physician). Table 2 shows the distribution of age and the test values in the study group. Plasma creatinine level is in- Symptoms cluded because it is important for the interpretation of 26,27 P-MMA levels. Symptoms possibly related to vitamin B12 deficiency were Using linear regression analysis we found strong posi- prevalent: 113 participants (28%) had more than 1 neu- tive associations between P-MMA level and age, P-tHcy rological symptom, 243 (60%) had at least 1 symptom level and age, and plasma creatinine level and age (PϽ.001 compatible with anemia, and 127 (32%) had at least 1 for all), but no association was found between the level gastrointestinal symptom. Figure 5 shows a weak as- of plasma cobalamins and age (P=.16). sociation between P-MMA concentration and the preva- Significant but not strong associations in the ex- lence of neurological symptoms, and a stronger associa- pected directions were found between the markers of vi- tion with age. When adjusting for age and sex, no tamin B12 deficiency: levels of P-MMA and plasma co- association was found between prestudy P-MMA levels balamins, r=−0.22; PϽ.001, log-transformed data; and and symptom scores of anemia (P=.68), neurological levels of P-MMA and P-tHcy, r=0.37; PϽ.001, log- (P=.56), or gastrointestinal (P=.76) symptoms or the to- transformed data. The associations were strong be- tal symptom score (P=.61). Neither did we find any as- tween levels of P-MMA as well as P-tHcy and plasma cre- sociations between follow-up levels of P-MMA, P-tHcy, atinine levels (Figure 3) and remained when controlled or plasma cobalamins and symptom scores (Table 3). for confounding by age. Adjustment for plasma creatinine level did not alter the Using linear regression analysis, P-MMA level was results (data not shown). weakly, but significantly, associated with blood hemo- Presuming that vitamin B12 deficiency is a likely di- globin level (PϽ.001) and erythrocyte mean cell vol- agnosis when levels of P-MMA and plasma cobalamins ume (P=.01) (Figure 4). However, we found no differ- are abnormal or levels of P-MMA and P-tHcy are abnor- ence in blood hemoglobin level (P=.94) or erythrocyte mal, we compared symptom scores in participants hav- mean cell volume (P=.96) comparing participants with ing 2 abnormal test results with those having 2 normal a 20% increase in P-MMA level with those with a 20% test results. Still, in these analyses, we found no associa- decrease in P-MMA level. Sixty participants (14%) had tion between the biochemical markers and symptom anemia, and a subgroup of 14 participants had blood he- scores. moglobin levels below the reference interval and eryth- We examined whether participants with an in- rocyte mean cell volumes greater than 95 fL. In this sub- crease in P-MMA concentration of more than 20% dif- group, no association was found among low blood fered from participants with a decrease of more than 20% hemoglobin levels, increased erythrocyte mean cell vol- between prestudy and follow-up. No association was umes, and levels of P-MMA (P=.93, linear regression ad- found between change in P-MMA level and the preva- justed for age, sex, and plasma creatinine level). lence of symptoms (linear regression adjusted for age and

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 5.00 12.00 A A

11.00 2.00

10.00 1.00

9.00 mol/L µ 0.50

8.00 P-MMA, Blood Hemoglobin, mmol/L 0.20 7.00

0.10 6.00 r = 0.29 r = –0.21 P<.001 P<.001

0.05 5.00

100.0 120 B B

50.0 110

20.0 100 mol/L µ

P-tHcy, P-tHcy, 10.0 90 Erythrocyte fL Mean Cell Volume, 5.0 80

r = 0.40 r = 0.12 P<.001 P = .01

2.0 70 50 (0.6) 100 (1.1) 200 (2.3) 500 (5.7) 0.05 0.10 0.20 0.50 1.00 2.00 5.00 Plasma Creatinine, µmol/L (mg/dL) P-MMA, µmol/L

Figure 3. Associations between plasma methylmalonic acid (P-MMA) (A) Figure 4. Associations between plasma methylmalonic acid (P-MMA) levels and plasma total homocysteine (P-tHcy) (B) levels and plasma creatinine and hematologic markers (A, blood hemoglobin level; B, erythrocyte mean level in 432 individuals not treated with cyanocobalamin. cell volume) in 432 individuals not treated with cyanocobalamin.

sex). Finally, no significant difference in prevalence of prestudy P-MMA level and the total Neurological Dis- symptoms was found between participants with P-MMA ability Score (r=0.10; P=.05, log-transformed data). No levels permanently greater than or equal to 0.40 µmol/L association was found between the follow-up P-MMA (n=60) and those whose levels were permanently less than level and the total Neurological Disability Score 0.40 µmol/L (n=256). (P=.64, log-transformed data). Furthermore, the Neu- Signs rological Disability Scores of participants having 2 abnormal test results did not differ from those having The maximum Neurological Disability Score was 112 2 normal test results. Participants with a P-MMA points. Eighty-three participants (21%) had a normal score increase of more than 20% or 50% did not have an of zero and 148 (37%) had a score of more than 10 points. increased Neurological Disability Score compared with Figure 6 shows the distribution of Neurological Dis- other participants. ability Scores at different levels of P-MMA and in differ- No associations were found between P-MMA ent age groups. The prevalence of a high Neurological level and nutritional state or neurological signs (Table Disability Score did not increase much with a higher level 3). Neither did we find any significant associations of P-MMA, whereas age and Neurological Disability Score between levels of P-tHcy or plasma cobalamins and were associated. the recorded signs. The results were essentially Using linear regression adjusted for age and sex we unchanged after adjustment for plasma creatinine level found a significant but weak association between the (data not shown).

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 VITAMIN SUPPLEMENTATION ≥4 Symptoms 2-3 Symptoms 0-1 Symptom

One hundred eighteen participants took vitamin supple- 100 ments daily typically containing 1 to 2 µg of cyanocobalamin and 200 µg of folic acid. We found a significant 80 inverse association between intake of vitamin supplements and P-tHcy level (P=.002, linear regression ad- 60 justed for age and sex). This association was not found for levels of P-MMA or plasma cobalamins. We did not 40 Participants, % find any association between estimates of vitamin B12 intake from food and levels of P-MMA, P-tHcy, and plasma 20 cobalamins (data not shown). 0 ≤0.28 0.29-0.39 ≥0.40 ≤54 55-74 ≥75 COMMENT P-MMA, µmol/L Age, y Figure 5. Neurological symptoms by level of plasma methylmalonic acid We studied 432 individuals not treated for vitamin B12 (P-MMA) and age group in 403 individuals not treated with deficiency despite an increased concentration of P-MMA. cyanocobalamin. We report a large variation in P-MMA levels over time. Furthermore, we found no association between the concentration of P-MMA and clinical manifestations related The concentration of P-MMA might be affected by to vitamin B12 deficiency. conditions other than vitamin B12 deficiency. Renal fail- 10,27,32 It is relatively easy to diagnose overt vitamin B12 de- ure is considered the most important condition, but 33 ficiency, but to diagnose mild vitamin B12 deficiency is intravascular volume depletion, changes in propionic difficult. If an elevated P-MMA level reflects a chronic acid–producing bacteria in the gut flora,8 pregnancy,34 or progressive condition, we would expect an increased and thyroid disease35 might also affect the P-MMA P-MMA level to be stable or to increase further over time level. In our study, renal failure was the most likely in individuals not treated with cyanocobalamin. The varia- confounder. However, our results remained essentially tion between the prestudy and follow-up P-MMA levels unchanged after controlling for plasma creatinine was considerable (coefficient of variation, 34%), indi- level. cating that the prestudy P-MMA level only contributes Participants were identified by the laboratory infor- little to the prediction of the P-MMA level at follow-up. mation system, implying some selection as they were seen In general, we did not find an increase in the P-MMA level by the general practitioner or hospitalized when the pre- measured 1.0 to 3.9 years after the initially increased level. study P-MMA level was measured. They thus represent An increased P-MMA level that normalizes on treat- individuals suspected of having vitamin B12 deficiency. ment with cyanocobalamin has been suggested as a di- We are confident that the 3-step procedure to identify agnostic test.2,10,11,28-30 Our results question this diagnos- individuals who had received cyanocobalamin treat- tic criterion because almost half the patients showed a ment was efficient and that the findings were not con- decrease of more than 20% in P-MMA concentration over founded by the effect of treatment. However, the asso- time without cyanocobalamin treatment. The average de- ciations between biochemical findings and clinical crease in P-MMA concentration can partly be explained manifestations might be affected by selection to treat- by regression toward the mean, but still the trend is re- ment of individuals with typical symptoms, leaving in- markable. dividuals with less pronounced symptoms for this study In the present study we examined the clinical cor- of the untreated. We cannot dismiss this selection bias; relates of abnormal levels of P-MMA, P-tHcy, and plasma however, in a previous study 36 of physicians’ reactions cobalamins. We used a structured interview to assess the to an increased concentration of P-MMA we found that symptoms, which allowed us to quantify neurological and only 22% of patients with an increased P-MMA level were gastrointestinal symptoms as well as symptoms of ane- selected for treatment and the remaining were not treated. mia. To assess neuromuscular dysfunction we chose the Treated patients did not differ from the untreated in clini- Neurological Disability Score, in which selected items from cal manifestations. the conventional neurological examination are scored.22 Based on our present results we disagree with authors This method is considered useful,31 but it has low sen- who suggest that P-MMA is a useful variable for screen- 37-39 sitivity and might not be as objective or reproducible as ing the elderly for vitamin B12 deficiency. Furthermore, desirable. we do not recommend use of an increased P-MMA con- Although the symptoms and signs related to vita- centration as the sole indicator for starting lifelong min B12 deficiency are not specific, we expected an as- cyanocobalamin treatment. If no other symptoms or signs sociation between the biochemical markers and the clini- indicate vitamin B12 deficiency, we suggest patients be cal manifestations. The associations found were followed up later rather than initiating treatment for vi- insignificant, weak, and in shifting directions. We found tamin B12 deficiency immediately. However, the rela- that age was a strong predictor for symptoms and signs, tively weak correlation between P-MMA and P-tHcy lev- whereas levels of P-MMA, P-tHcy, and plasma cobala- els might well be explained by the fact that the P-tHcy mins did not add further to the prediction of clinical mani- level increases also in patients with folate or vitamin B6 festations. deficiency. Hyperhomocysteinemia has recently been

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Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 3. Associations Between the Biochemical Markers and Symptoms and Signs of Vitamin B12 Deficiency, Adjusted for Age and Sex, in 403 Participants Who Underwent Clinical Examination*

P-MMA P-tHcy Plasma Cobalamins Symptoms Symptoms of anemia† .31‡ .15 Ͼ.99 Neurological symptoms† .73 .50 .57 Gastrointestinal symptoms† .13‡ .01 .87‡ Total symptom score† .81‡ .11 .78 Signs Neurological Disability Score† .64‡ .62 .39 Nutritional state§ 0.87 (0.43-1.76) 1.84 (0.73-4.68) 0.83 (0.35-1.94) Insecure “finger-nose” test§ 0.78 (0.47-1.29) 1.08 (0.55-2.12) 1.67 (0.93-3.01) Insecure “heel-knee-shin” test§ 1.03 (0.62-1.71) 1.23 (0.61-2.47) 1.09 (0.60-1.98) Dysdiadochokinesis§ 0.43 (0.14-1.29) 0.99 (0.27-3.60) 1.23 (0.42-3.60) Present Romberg sign§ 0.67 (0.40-1.13) 1.04 (0.53-2.07) 1.03 (0.57-1.87) Abnormal gait§ 1.09 (0.65-1.83) 1.96 (0.97-3.99) 0.81 (0.45-1.47)

*P-MMA indicates plasma methylmalonic acid; P-tHcy, plasma total homocysteine. †Linear regression analysis. Data are given as P values. ‡The direction of the association was opposite that expected. §Logistic regression analysis. Data are given as odds ratios per unit change in the log-transformed biochemical markers (95% confidence intervals).

Foundation; the Hans and Nora Buchard Foundation; grant NDS ≥21 NDS 11-20 NDS 1-10 NDS 0 (Normal) BMH4-98-3549 from EU Biomed; The Gangsted Foundation; 100 the L.F. Foghts Foundation; The Novo Nordisk Foundation; and the Oda and Hans Svenningsen Foundation. 80 We thank Erik Kjærsgaard Hansen, MSc, and Jens Bar- fred Jensen, Department of Clinical Biochemistry, Aarhus 60 Kommunehospital/Skejby Sygehus, Aarhus University Hospi- tal, for help concerning data from the laboratory information 40 system; Birgitte Holm Andersen, MSc, and Jørgen Nørskov Participants, % Nielsen, MSc, National Health Insurance, for information on 20 prescriptionsofcyanocobalaminpreparations;SvendJuul,MD, Department of Epidemiology and Social Medicine, Aarhus 0 ≤0.28 0.29-0.39 ≥0.40 ≤54 55-74 ≥75 University, for statistical assistance; Birgitte Horst Andreasen, P-MMA, µmol/L Age, y RN, Department of Hematology, Aarhus University Hospital, for help with the clinical examinations; and the staff of the Figure 6. Neurological Disability Scores (NDS) by level of plasma methylmalonic acid (P-MMA) and age group in 403 individuals not treated Department of Clinical Biochemistry, Aarhus University Hos- with cyanocobalamin. pital, for performing the biochemical analyses. Corresponding author and reprints: Anne-Mette Hvas, MD, Department of Hematology, Aarhus University Hos- 40 linked to cardiovascular disease, but it is still dis- pital, Aarhus Amtssygehus, Tage Hansens Gade 2, 8000 cussed whether treatment or prevention with B vita- Aarhus C, Denmark (e-mail: [email protected]). mins will reduce the cardiovascular risk.41 The lack of association between clinical manifesta- REFERENCES tions and biochemical indices of vitamin B12 deficiency might be attributable to the high prevalence of symp- 1. Carmel R. Pernicious anemia: the expected findings of very low serum cobala- toms of anemia and neurological abnormalities in the el- min levels, anemia, and macrocytosis are often lacking. Arch Intern Med. 1988; derly. We expect that individuals with clinical manifes- 148:1712-1714. tations and abnormal values for one or more of the 2. Lindenbaum J, Healton EB, Savage DG, et al. Neuropsychiatric disorders caused biochemical markers would benefit from treatment with by cobalamin deficiency in the absence of anemia or macrocytosis. N Engl J Med. 1988;318:1720-1728. cyanocobalamin, but at present we do not know whether 3. Carmel R, Gott PS, Waters CH, et al. The frequently low cobalamin levels in de- this is the case. A randomized clinical study is needed to mentia usually signify treatable metabolic, neurologic and electrophysiologic ab- answer this important question. normalities. Eur J Haematol. 1995;54:245-253. 4. Healton EB, Savage DG, Brust JC, Garrett TJ, Lindenbaum J. Neurologic aspects Accepted for publication November 7, 2000. of cobalamin deficiency. Medicine (Baltimore). 1991;70:229-245. 5. Martin DC, Francis J, Protetch J, Huff J. Time dependency of cognitive recovery with This work was supported in part by a grant from The cobalamin replacement: report of a pilot study. J Am Geriatr Soc. 1992;40:168-172. Health Found of “danmark’s” Sygeforsikring, and grant 6. Carmel R, Karnaze DS. The deoxyuridine suppression test identifies subtle co- 9802749 from The Danish Medical Research Council, Co- balamin deficiency in patients without typical megaloblastic anemia. JAMA. 1985; penhagen; The Institute of Experimental Clinical Research, 253:1284-1287. 7. Carmel R, Sinow RM, Karnaze DS. Atypical cobalamin deficiency: subtle bio- AarhusUniversity,Aarhus,Denmark;theC.C.KlestrupFoun- chemical evidence of deficiency is commonly demonstrable in patients without dation; the Johannes and Ella Fogh-Nielsen Foundation; the megaloblastic anemia and is often associated with protein-bound cobalamin mal- JacobandOlgaMadsenFoundation;theE.DanielsenandWife’s absorption. J Lab Clin Med. 1987;109:454-463.

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