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An Adolescent with Increased Plasma Methylmalonic Acid and Total Homocysteine.


An 18-year-old male patient was transferred to our hospital for evaluation of recent-onset generalized weakness, difficulty walking, and a 3-week history of progressive numbness in his fingertips and distal extremities. The patient's mother reported that she had experienced 4 similar episodes of lower-body weakness at the age of 16 years that had self-resolved. In addition, the patient's brother was hospitalized for a similar episode of lower-body weakness that had also self-resolved.

The patient's neurologic exam was notable for difficulty walking, with an unsteady, wide-based gait. He could not walk on toes or heels without assistance; otherwise he had full strength on manual muscle testing. Sensory exam revealed decreased appreciation of sensation to touch, temperature, vibration and proprioception (position sense) from feet proximally to knees and fingertips to palms.

Laboratory findings were as follows: plasma methylmalonic acid (MMA) [5] was highly increased: 17.4 [micro]mol/L (reference interval: 0-0.29 [micro]mol/L); total homocysteine was highly increased: 125 [micro]mol/L (reference interval: <12.5 [micro]mol/L); vitamin [B.sub.12] was low: 188 pg/mL (200-1100 pg/mL); and liver enzymes were slightly increased: alanine transaminase (ALT): 54 U/L (reference interval: 3-35 U/L); aspartate transaminase (AST): 48 U/L (reference interval: 15-46 U/L). All other blood test results were within reference intervals except for a slightly increased red cell distribution width (RDW)-CV, 18.0% (reference interval: 11.5%-14.5%) and very slightly decreased hematocrit (HCT), 38.4% (reference interval: 39%-53%). There was no laboratory evidence of megaloblastic anemia.

The patient was treated with 1 intramuscular injection of cyanocobalamin and referred to an adolescent clinic for psychosocial evaluation, but he then left the hospital against medical advice before further treatment.

Eight months later, he presented with similar findings with loss of lower-extremity proprioception and vibratory sense, spastic diplegia with foot drop, and wide-based ataxic gait. He was admitted to the hospital again due to worsening gait progressing to a nonambulatory status. His laboratory findings were as follows: MMA was highly increased, 8.6 [micro]mol/L (reference interval: 0-0.29 [micro]mol/L); total homocysteine was highly increased: 116 [micro]mol/L (reference interval <12.5 [micro]mol/L); vitamin [B.sub.12] was low: 149 pg/mL (reference interval: 200-1100 pg/ mL); folate >24.0 ng/mL (reference interval: 2-20 ng/ mL). His RDW-CV was very slightly increased, 14.8% (reference interval: 11.5%--14.5%). All other blood tests were essentially within reference intervals. MRI of brain and spine did not show specific findings. Electromyography (EMG) was consistent with mixed axonal demyelinating polyneuropathy.


Vitamin [B.sub.12] [cobalamin (Cbl)] plays an essential role both in the conversion of methylmalonyl-CoA to succinyl-CoA and in the synthesis of methionine (Met) from homocysteine (Hcy) (Fig. 1). Two enzymes are involved in this pathway, methylmalonyl-CoA mutase and methionine synthase. Adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) are cofactors for these 2 enzymes. The disorders of intracellular cobalamin metabolism result from deficient synthesis of AdoCbl and/or MeCbl derived from vitamin [B.sub.12]. Simultaneous increases in both MMA and total Hcy (tHcy) are seen in inherited intracellular cobalamin defects, vitamin [B.sub.12] deficiency, Imerslund-Grasbeck syndrome, and transcobalamin II deficiency. Patients with inherited intracellular cobalamin defects usually present with higher concentrations of MMA and/or tHcy than patients with vitamin [B.sub.12] deficiency. Subtypes CblC, CblD, CblF, and CblJ affect both AdoCbl and MeCbl and cause combined increases of MMA and tHcy. CblC is the most common among all the subtypes of inherited intracellular cobalamin defects, the age of onset ranges from prenatal to adolescent and to adulthood, although the infantile presentation is the most frequently recognized. To diagnose these disorders of intracellular cobalamin metabolism, and to follow up abnormal newborn screenings that demonstrated the presence of increased C3 propionylcarnitine or decreased methionine, a fast, sensitive, and simple method for the simultaneous detection of plasma tHcy, MMA, Met, and 2-methylcitric acid (2MCA) using LC-MS was implemented in the Biochemical Genetics and Special Chemistry laboratory at Children's Hospital Los Angeles (1).

Vitamin [B.sub.12] is necessary for DNA synthesis and methylation reactions. Its deficiency is associated with hematologic, neurologic, and psychiatric manifestations. Neurologic sequelae from vitamin [B.sub.12] deficiency include paresthesias, peripheral neuropathy, and demyelination of the corticospinal tract and dorsal columns [subacute combined degeneration of the spinal cord (SCD)]. Vitamin [B.sub.12] deficiency is usually caused by a restrictive vegetarian diet, malnutrition, malabsorption syndromes, pernicious anemia (PA) caused by gastric intrinsic factor deficiency, congenital disease, gastrectomy, or other gastrointestinal tract diseases. However, it may be also induced by the recreational use of nitrous oxide ([N.sub.2]O), an often forgotten and important link to vitamin [B.sub.12] deficiency in young, healthy individuals.

[N.sub.2]O, commonly known as laughing gas, is routinely used for its anesthetic and analgesic effects, particularly during dental procedures. It is also used as an oxidizer in rockets and motor racing to increase the power output of engines. [N.sub.2]O can also be obtained in grocery stores in the form of whipped cream canisters and small bulbs called "whippets," which contain [N.sub.2]O (2). A simple Google search shows the variety ofnitrous oxide products that are readily available. Since [N.sub.2]O is commonly administered as anesthesia, a common misconception is that it is safe to use recreationally. The original research from the largest 2014 Global Drug Survey (GDS) (n = 74864) confirms [N.sub.2]O as a very common drug ofabuse, in particular in the UK and US (38.6% and 29.4% lifetime prevalence) (3). It appears that [N.sub.2]O is currently being abused across multiple populations and represents a significant public health problem (4). Yet [N.sub.2]O toxicity is often absent from differential diagnoses.

Oxidation of the cobalt ion by [N.sub.2]O prevents methylcobalamin from acting as a coenzyme in the production of methionine and subsequently S-adenosyl-L-methionine (SAM), which is necessary for methylation of myelin sheath phospholipids (5). This results in demyelination of the spinal cord. Additionally, there is impaired ability to convert 5-methyltetrahydrofolate to tetrahydrofolate, impacting DNA synthesis. Moreover, inactivated [B.sub.12] renders methylmalonyl-CoA mutase unable to effectively convert methylmalonyl-CoA to succinyl-CoA, increasing plasma concentrations of MMA (4) (Fig. 1).

[N.sub.2]O-induced neuropathy was initially recognized by Layzer et al. in 1978 (6). In that study, the common initial symptoms were numbness of the distal limbs and imbalance complicated by weakness in the lower extremities, with some patients becoming too unsteady to walk without assistance. The numbness sometimes travelled upwards from the feet and also caused clumsiness in the hands. The majority of cases were diagnosed with neurologic sequelae such as myeloneuropathy and SCD and were often accompanied by neuroimaging changes (4). In other cases patients have presented with generalized weakness, fatigue, and unexplained psychiatric abnormalities such as psychotic behavior, acute loss of memory, and mood disorders (7).

Although vitamin [B.sub.12] deficiency is known to cause megaloblastic anemia, our patient had an MCV within the reference interval, a slight increase of RDW-CV of 18.0%, and hemoglobin (Hb) within the reference interval at 13.6 g/dL. In fact, most cases of SCD caused by [N.sub.2]O misuse reported in the literature do not exhibit megaloblastic anemia. As proposed by Alt et al. (2), it is likely that adequate folic acid dietary intake works in the DNA synthetic pathway via a different route, bypassing the methionine synthase reaction. Therefore, patients with adequate folic acid intake may not develop the hematologic effects of vitamin [B.sub.12] deficiency despite overt neurologic dysfunction (2). In our case, the folate concentration was >24 ng/mL when the patient was admitted to the hospital a second time, which was 8 months after the first episode.

Laboratorians and clinicians should be aware of the increasing incidence of [N.sub.2]O abuse and how to diagnose it. In our case, a LC-MS/MS method for the simultaneous detection of plasma tHcy, MMA, Met, and 2MCA was implemented. Simultaneous increase in both MMA and tHcy was recognized. The concentration of MMA was 60 times the upper limit of the reference interval, and the increase in tHcy was 10 times the upper limit of the reference interval. Concentrations of MMA and tHcy were similar to those observed for several patients with inherited cobalamin C defect followed in our laboratory; therefore, the possibility of an intracellular cobalamin defect was initially raised and communicated to the attending pediatric neurologist. The diagnosis of vitamin [B.sub.12] deficiency due to [N.sub.2]O abuse was reached by combining the clinical presentation, laboratory values, and medical and family histories. In this case, the patient reported that he had been inhaling significant quantities of [N.sub.2]O on a daily basis for an undetermined period of time. The vitamin [B.sub.12] concentrations in this patient were slightly reduced: 188 pg/mL and 149 pg/mL 8 months later.

The ensuing neurological damage from recreational abuse of [N.sub.2]O can be transient or permanent depending on the severity of [N.sub.2]O usage. Some studies found that recovery from [N.sub.2]O neuropathy may be slow and incomplete: despite high-dose vitamin [B.sub.12] replacement (8). In our case, the patient was again admitted with similar symptoms because he stopped vitamin [B.sub.12] supplementation and reinhaled [N.sub.2]O. His brain and spine MRI did not show specific findings on the second hospitalization, but his EMG was consistent with mixed axonal demyelinating polyneuropathy. It is important to raise clinical awareness of [N.sub.2]O-induced SCD of the spinal cord, and it should be considered in the differential diagnosis when a patient presents with progressive myelopathy and/or mental status changes. Patients, especially adolescents, should be queried for [N.sub.2]O exposure and/or abuse in cases of vitamin cobalamin deficiency of unknown origin, and physicians should request laboratory tests for MMA, tHcy, and vitamin [B.sub.12].

In summary, we report an 18-year-old male patient who presented with myelopathy without megaloblastic anemia with highly increased MMA, tHcy, and slightly reduced concentrations of vitamin [B.sub.12] from vitamin [B.sub.12] deficiency caused by [N.sub.2]O abuse. With the increasing incidence of [N.sub.2]O misuse in adolescents and young adults, this case highlights the importance of considering [N.sub.2]O toxicity-induced SCD of the spinal cord in the differential diagnosis of patients with undifferentiated neurological symptoms.


1. Describe the metabolites that are increased in inherited intracellular vitamin [B.sub.12] (cobalamin) defects.

2. Explain the clinical and biochemical findings in vitamin [B.sub.12] deficiency.

3. List potential causes of vitamin [B.sub.12] deficiency.


* [N.sub.2]O abuse is not uncommon among adolescents and young adults.

* [N.sub.2]O inhalation should be considered as a potential cause of vitamin [B.sub.12] deficiency.

* The neurological damage can be transient or permanent, depending on the severity of [N.sub.2]O usage.

* Measurement of MMA, tHcy concentrations, and vitamin [B.sub.12] can be used to make the diagnosis of vitamin [B.sub.12] deficiency in the presence of reference or low-reference vitamin [B.sub.12] concentration.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: C. Francisco, Children's Hospital Los Angeles.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Patents: None declared.


(1.) Fu X, Xu YK, Chan P, Pattengale PK. Simple, fast, and simultaneous detection of plasma total homocysteine, methylmalonic acid, methionine, and 2-methylcitric acid using liquid chromatography and mass spectrometry (LC/MS/MS). JIMD Rep 2013;10:69-78.

(2.) Alt RS, Morrissey RP, Gang MA, Hoffman RS, Schaumburg HH. Severe myeloneuropathy from acute high-dose nitrous oxide ([N.sub.2]O) abuse. J Emerg Med 2011;41:378- 80.

(3.) Kaar SJ, Ferris J, Waldron J, Devaney M, Ramsey J, Winstock AR. Up: The rise of nitrous oxide abuse. An international survey of contemporary nitrous oxide use. J Psychopharmacol 2016;30:395-401.

(4.) Garakani A, Jaffe RJ, Savla D, Welch AK, Protin CA, Bryson EO, McDowell DM. Neurologic, psychiatric, and other medical manifestations of nitrous oxide abuse: a systematic review of the case literature. Am J Addict 2016;25:358-69.

(5.) Pema PJ, Horak HA, Wyatt RH. Myelopathy caused by nitrous oxide toxicity. AJNR Am J Neuroradiol 1998;19:894-6.

(6.) Layzer RB, Fishman RA, Schafer JA. Neuropathy following abuse of nitrous- oxide ([N.sub.2]O). Neurology1978;28:504-6.

(7.) Cousaert C, Heylens G, Audenaert K. Laughing gas abuse is no joke. An overview of the implications for psychiatric practice. Clin Neurol Neurosurg 2013;115:859- 62.

(8.) Thompson AG, Leite MI, Lunn MP, Bennett DL. Whippits, nitrous oxide and the dangers of legal highs. Pract Neurol 2015;15:207-9.

Xiaowei Fu, [1,2] * Carla Francisco, [3] Paul Pattengale, [1,2] Maurice R. O'Gorman, [1,2,4] and Wendy G. Mitchell [3]

[1] Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA; [2] Department of Pathology and Laboratory Medicine, Children's Hospital of Los Angeles, Los Angeles, CA; [3] Division of Neurology, Department of Pediatrics, Children's Hospital of Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA; [4] Department of Pediatrics, Keck School of Medicine, University of

Southern California, Los Angeles, CA.

* Address correspondence to this author at: Department of Clinical Pathology, Keck School of Medicine, University of Southern California, Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, 4650 Sunset Blvd., MS#32, Los Angeles, CA 90027. Fax 323-361-6157; e-mail

Received May 18,2016; accepted October 3,2016.

DOI: 10.1373/clinchem.2016.260695

[C] 2016 American Association for Clinical Chemistry

[5] Nonstandard abbreviations: MMA, methylmalonic acid; ALT, alanine transaminase; AST, aspartate transaminase; RDW, red cell distribution width; HCT, hematocrit; EMG, electromyography; Cbl, cobalamin; Met, methionine; Hcy, homocysteine; AdoCbl, adenosylcobalamin; MeCbl, methylcobalamin; tHcy, total Hcy; 2MCA, 2-methylcitric acid; SCD, subacute combined degeneration of spinal cord; PA, pernicious anemia; [N.sub.2]O, nitrous oxide; GDS, Global Drug Survey; SAM, 5- adenosykmethionine; Hb, hemoglobin.

Caption: Fig. 1. The metabolic pathways enhanced by cobalamin and inherited disorders of cobalamin. 5-deoxyadenosylcobalamin (AdoCbl) is required by methylmalonyl-CoA mutase, which catalyzes the conversion of L- methylmalonyl-CoA to succinyl-CoA. Succinyl-CoA then enters the citric acid cycle. Methionine synthase is a vitamin [B.sub.12]-dependent enzyme that catalyzes the formation of Met from Hcy using 5-methyltetrahydrofolate (5-methyl THF), a folate derivative, as a methyl donor. Met, in the form of SAM, is required for most biological methylation reactions, including DNA methylation. Methyl-Cbl, methyl- cobalamin; SAH, S-adenosyl-L-homocysteine; X, methyl acceptor; C[H.sub.3]-X, methylated product; Pi, phosphate; PPi, pyrophosphate.
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Title Annotation:Clinical Case Study
Author:Fu, Xiaowei; Francisco, Carla; Pattengale, Paul; O'Gorman, Maurice R.; Mitchell, Wendy G.
Publication:Clinical Chemistry
Date:Jun 1, 2017
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