Clinical relevance of testing for metabolic vitamin B12 deficiency in patients with polyneuropathy

Figures & data

Table 1. Clinical and laboratory characteristics of 331 patients with a polyneuropathy.

Age, mean (SD)		63.0 (11.4)
Men, n (%)		235 (71.0)
Symptom duration in months, median (IQR)		36 (55.7)
Etiology of polyneuropathy n (%)
	Diabetes	22 (6.6)
	Hereditary	51 (15.4)
	Immune-mediated^a	30 (9.1)
	Toxic (alcohol, medication)	26 (7.9)
	Vitamin deficiency (B12 or B1)	20 (6.0)
	Systemic disorder^b	12 (3.6)
	Metabolic (uremic, thyroid)	11 (3.3)
	Multifactorial	25 (7.6)
	CAP	134 (40.5)
Vitamin B12 pmol/L, median (IQR)		217 (110)
MMA μmol/L, median (IQR)		0.19 (0.1)
Hcy μmol/L, median (IQR)		11.8 (4.7)
Hemoglobin mmol/L, mean (SD)		9.0 (0.8)
MCV fL, mean (SD)		90.5 (4.6)

IQR, Interquartile range; CAP, cryptogenic axonal polyneuropathy; MMA, methylmalonic acid; Hcy, homocysteine; MCV, mean corpuscular volume.

^a Chronic inflammatory demyelinating polyneuropathy, Guillain Barre Syndrome, M-protein associated.

^b Sarcoidosis, amyloidosis, vasculitis, m. Sjögren, Rheumatoid arthritis.

Figure 1. Metabolite levels and vitamin B12 levels. (a) Dot plot of vitamin B12 and methylmalonic acid levels with trend line, Pearson correlation after logarithm transformation: −0.39 (95% CI −0.48- −0.29). (b) Dot plot of vitamin B12 and homocysteine levels with trend line, Pearson correlation after logarithm transformation: −0.32 (95% CI −0.41- −0.22).

Table 2. Prevalence of elevated metabolites per vitamin B12 category.

Vitamin B12 categories, pmol/L	<148 (n=51)	148–200 (n = 89)	200–250 (n = 80)	250–300 (n = 43)	>300 (n = 68)	Total (n = 331)	p-Value^a
Elevated MMA, n (%)	20 (39.2)	19 (21.3)	9 (11.3)	2 (4.7)	2 (2.9)	52 (15.7)	<0.01
Elevated Hcy, n (%)	23 (45.1)	30 (33.7)	24 (30.0)	5 (11.6)	12 (17.6)	94 (28.4)	<0.01
Elevated MMA and Hcy, n (%)	11 (21.5)	10 (11.2)	4 (5.0)	0 (0)	1 (1.5)	26 (7.9)	<0.01
Elevated MMA or Hcy, n (%)	32 (62.3)	39 (43.8)	29 (36.2)	7 (16.3)	13 (19.1)	120 (36.3)	<0.01
Normal MMA and Hcy, n (%)	19 (37.3)	50 (56.2)	51 (63.7)	36 (83.7)	55 (80.9)	211 (63.7)	<0.01

MMA, methylmalonic acid; Hcy, homocysteine.

^a Difference between vitamin B12 categories using χ².

Figure 2. Probability of elevated metabolites as function of vitamin B12 levels; predicted probabilities estimated using a logistic regression model with elevated metabolites as dependent variable and vitamin B12 level as covariate. Abbreviations: MMA, methylmalonic acid; Hcy, homocysteine.

Table 3. Logistic regression for Vitamin B12 as covariate, after excluding vitamin B12 < 148 pmol/L (200 pg/mL), n = 285.

	Vitamin B12 threshold, pmol/L	Sensitivity	Specificity	Prevalence of elevated metabolite(s), n (%)	NND	Missed cases of elevated metabolites, n
MMA > 0.29 μmol/L: AUC 0.72
Best trade-off	213	0.69	0.66	22 (7.9%)	4.8	10
95% sensitivity	304	0.97	0.26	30 (10.7%)	7.1	1
90% sensitivity	264	0.91	0.38	28 (10.0%)	6.4	3
MMA > 0.29 μmol/L and/or Hcy > 14 μmol/L: AUC 0.65
Best trade-off	257	0.80	0.46	70 (25.0%)	2.5	18
Sensitivity 95%	368	0.96	0.10	83 (29.6%)	3.0	4
Sensitivity 90%	327	0.91	0.23	79 (28.2%)	2.9	8
Hcy > 14 μmol/L: AUC 0.61
Best trade-off	257	0.79	0.44	56 (20.0%)	3.1	15
Sensitivity 95%	374	0.96	0.10	67 (23.9%)	3.8	3
Sensitivity 90%	329	0.90	0.21	63 (22.5%)	3.6	7

NND, number needed to diagnose; MMA, methylmalonic acid; Hcy, homocysteine.

Table 4. Comparison of clinical phenotype and laboratory results between patients with absolute, metabolic and no vitamin B12 deficiency.

	All patients, n = 331	Absolute B12 deficiency^a, n = 52	Metabolic B12 deficiency^b, n = 30	No B12 deficiency^c, n = 250	p-Value^d
Clinical phenotype n (%)
Sensorimotor	204 (61.6)	28 (45.1)	16 (46.7)	117 (46.8)
Pure sensory	127 (38.4)	23 (54.9)	14 (53.3)	133 (53.2)	0.98^e
Onset of symptoms, n (%):
Arms	16 (4.8)	1 (2.0)	3 (10.0)	12 (4.9)	0.26^f
Arms and legs	22 (6.6)	2 (3.9)	2 (6.7)	18 (7.2)	0.74^f
Legs	288 (87.0)	47 (92.2)	25 (83.3)	216 (86.4)	0.42^e
Unknown	5 (1.5)	1 (2.0)	0 (0)	4 (1.6)
MMA μmol/L, median (IQR)	0.19 (0.1)	0.24 (0.16)	0.36 (0.18)	0.17 (0.08)	<0.01^g
Hcy μmol/L, median (IQR)	11.8 (4.7)	13.9 (5.9)	14 (4.1)	11.25 (4.4)	<0.01^h
Hemoglobin mmol/L, mean (SD)	9.0 (0.8)	8.8 (0.9)	9.3 (1.25)	9 (1.0)	0.20^i
MCV fL, mean (SD)	90.5 (4.6)	91 (6.)	89.5 (4.7)	90 (5.0)	0.05

^a Vitamin B12 >148 pmol/L, ^bVitamin B12 148–304 pmol/L and methylmalonic acid >0.29 μmol/L, ^cVitamin B12 > 148 pmol/L and normal methylmalonic acid or Vitamin B12 > 304pmol/L, ^dDifference between vitamin B12 categories, ^eχ²,^fFisher exact test, ^gone-way Anova test, all groups differ when applying Tukey multiple pairwise-comparisons, ^hone-way Anova test, no deficiency differs from both groups with deficiency when applying Tukey multiple pairwise-comparisons, ⁱone-way Anova test, no deficiency differs from absolute deficiency when applying Tukey multiple pairwise-comparisons.

Table 5. Effect of vitamin B12 supplementation and prevalence of elevated metabolites, n (%).

Supplementation effect	Deteriorated	Stable	Improved	p-value^a
All patients n = 42
MMA elevated	13 (31.0)	6 (14.3)	3 (7.1)	0.76
Hcy elevated	13 (31.0)	6 (14.3)	3 (7.1)	0.76
Both MMA and Hcy elevated	6 (14.3)	4 (9.5)	2 (4.8)	0.65
Both normal	4 (9.5)	2 (4.8)	4 (9.5)	0.22
Total	24 (57.1)	10 (23.8)	8 (19.0)
Vitamin B12 < 148 pmol/L (200 pg/mL), n = 23
MMA elevated	8 (34.8)	3 (13.0)	1 (4.3)	0.26
Hcy elevated	7 (30.4)	3 (13.0)	2 (8.7)	0.86
Both MMA and Hcy elevated	4 (17.4)	1 (4.3)	1 (4.3)	0.84
Both normal	1 (4.3)	1 (4.3)	3 (13.0)	0.06
Total	12 (52.2)	6 (26.1)	5 (21.7)
Vitamin B12 ≥ 148 pmol/L (200 pg/mL), n = 19
MMA elevated	5 (26.3)	3 (15.7)	2 (10.5)	0.55
Hcy elevated	6 (31.6)	3 (15.7)	1 (5.3)	0.67
Both MMA and Hcy elevated	2 (10.5)	3 (15.7)	1 (5.3)	0.06
Both normal	3 (15.7)	1 (5.3)	1 (5.3)	1
Total	12 (63.2)	4 (21.1)	3 (15.8)

MMA, methylmalonic acid; Hcy, homocysteine.

^a Difference between supplementation effect groups using Fisher exact test.

Table 6. Results of patients with CAP compared to patients with polyneuropathy with a known underlying etiology (non-CAP).

	CAP	Non-CAP	p-value
MMA elevated, n (%)	15 (11.7)	17 (11.2)	1.0^b
Hcy elevated, n (%)	26 (20.3)	45 (29.6)	0.10^b
Both MMA and Hcy elevated, n (%)	5 (3.9)	10 (6.6)	0.47^b
Both normal, n (%)	92 (71.9)	100 (65.8)	0.34^b
Metabolic B12 deficiency^a, n (%)	15 (88.3)	15 (90.1)	0.76^b
Vitamin B12, pmol/L (median, IQR)	232 (99.8)	239.5 (124.5)	0.63^c
MMA, μmol/L (median, IQR)	0.19 (0.10)	0.19 (0.09)	0.34^c
Hcy, μmol/L (median, IQR)	11.5 (3.5)	11.7 (5.1)	0.76^c
Effect of supplementation
Improvement, n (%)	1 (10.0)	2 (22.2)	0.41^d
Stable, n (%)	2 (10.0)	2 (22.2)	0.64^d
Deterioration, n (%)	7 (70.0)	5 (55.6)	0.55^d

CAP, cryptogenic axonal polyneuropathy.

^a Vitamin B12 > 148 pmol/l and elevated MMA (>0.29 μmol/L).; ^bChi-squared test; ^cStudents t-test after log transformation; ^dFisher exact test.

Figure 3. ROC curves for elevated metabolites as dependent and vitamin B12 levels as covariate. Methylmalonic acid >0.29 μmol/L as dependent. Methylmalonic acid >0.29 μmol/L or homocysteine >14 μmol/L as dependent. Homocysteine >14 μmol/L as dependent. Abbreviations: MMA, methylmalonic acid; Hcy, homocysteine.

Figure 4. Flow chart for diagnosing vitamin B12 deficiency in patients with polyneuropathy. Abbreviations: LLN, lower limit of normal; MMA, methylmalonic acid.

Data availability statement

Pseudonimized data are available upon reasonable request.