**Background:** Nicotinamide adenine dinucleotide (NAD(H)) is a coenzyme essential for energy metabolism, DNA repair, gene expression, and stress response. NAD(H) is synthesized primarily through the salvage pathway, where nicotinamide is converted to 1-methylnicotinamide by nicotinamide N-methyltransferase (NNMT) or to nicotinamide mononucleotide by NAMPT. Aberrant NNMT expression has been linked to obesity, type 2 diabetes, cancers, neurodegenerative diseases, and psychiatric disorders, but no prior report has described critically decreased NNMT function manifesting in adulthood.
**Methods:** The patient was a 20-year-old male who developed flu-like symptoms at age 18 that progressed over approximately 4 years to a non-verbal, wheelchair-dependent state requiring a gastric feeding tube, tracheostomy with oxygen, and round-the-clock care. Extensive prior workup including brain MRI (bilateral putamen T2 hyperintensities), EEG (encephalopathy), CSF analysis (elevated total Tau protein at 2936 ng/mL, positive 14-3-3 protein), whole exome sequencing, mitochondrial DNA sequencing, and whole genome sequencing of the patient and parents did not yield a definitive diagnosis. A prior metabolic panel showed absence of 1-methylnicotinamide and low N1-methyl-2-pyridone-5-carboxamide. For the present study, plasma samples were collected under three conditions: (1) normal fed conditions (baseline, collected 3 months prior), (2) immediately after a 4-hour fast, and (3) 8 hours after a 500 mg liquid nicotinamide bolus via gastric feeding tube. Global untargeted metabolic profiling was performed using ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) across four chromatography methods. Compounds were identified by comparing mass-to-charge ratio, retention time, and fragmentation spectra to a library of standards. Raw peak values were log-transformed and converted to Z-scores relative to a healthy reference population. Analytical precision was confirmed with 6 technical replicates of plasma (872 metabolites detected in all replicates; mean RSD 9.04%, median RSD 3.7%; internal standards mean and median RSD 5.1% and 4.1%, respectively).
**Key Results:** The patient's nicotinamide levels were in the 100th percentile at all three time points due to high-dose supplementation. Baseline and fasting 1-methylnicotinamide levels were below the 2.5th percentile, indicating decreased NNMT function. The nicotinamide challenge restored 1-methylnicotinamide to normal levels, demonstrating inducible NNMT activity. Nicotinamide riboside was normal at baseline (40th percentile) and rose only slightly post-challenge (44th percentile). Tryptophan, kynurenine, and quinolinate in the de novo NAD(H) pathway were within low normal range at all time points. Methionine was absent at baseline and after fasting but was restored to the 7th percentile post-challenge. Cystathionine was normal at all time points, while cysteine was significantly elevated (p < 0.05) at all time points. In purine metabolism, inosine and hypoxanthine were above the 97.5th percentile at baseline and after fasting; hypoxanthine returned to normal post-challenge. Xanthine fell from the 47th to the 19th percentile post-challenge. Several n-3 PUFAs (stearidonate, docosapentaenoate, eicosapentaenoate) were above normal at baseline and after fasting and remained elevated post-challenge. Cortisol and cortisone increased significantly post-challenge but remained within normal range. The patient died in March 2022, approximately 4 years after illness onset, with tachycardia (150-170 bpm) giving way to cardiac arrest.
**Clinical Implications:** This is the first reported case of critically decreased NNMT function manifesting in adulthood and associated with neurodegenerative disease. The defect was inducible by high-dose nicotinamide after fasting, but metabolic rescue did not impact clinical status, possibly due to advanced disease stage or additional unidentified metabolic defects. The study demonstrates that untargeted metabolomics can reveal deep phenotypic information in rare metabolic diseases even when genomic sequencing is inconclusive. Limitations include the lack of muscle tissue and CSF metabolic profiling, and the inability to determine whether the NNMT defect was inborn or acquired in the absence of a conclusive genetic cause.