# Argininemia: Pathophysiology and Novel Methods for Evaluation of the Disease
## 4 Novel Methods of Evaluation  
### Current Methods of Diagnosis and Treatment  
Diagnosing ARG1-D involves a combination of high clinical suspicion and specific biochemical findings. Confirmatory methods include genetic testing or the inability to detect red blood cell arginase enzyme activity, procedures also followed in the context of newborn screening (NBS) [22; 67; 68]. Laboratory methods used for diagnosis encompass measuring arginine and other amino acid levels, as well as metabolites and even ammonia levels [3]. Most frequently arginine levels are measured in plasma using mass spectrometry, but also other types of samples, such as CSF, urine, and leukocytes can be used. Red blood cell arginase activity is evaluated in fresh or dried blood samples [67]. In the case of arginine, arginine levels can be as high as four times the normal (>300 umol/L) and red blood cell arginase activity less than 1% of normal levels [3,22]. Assessing the levels of other amino acids, such as ornithine, glutamine, phenylalanine, aspartic acid, lysine, and threonine--that are typically abnormal in hyperarginine patients--in plasma or erythrocytes can help validate the diagnosis. Metabolites like orotic acid, guanidino compounds, glutathione, prealbumin, and pyrimidine can also be measured in adequate samples, such as plasma, serum, erythrocytes, or urine, as reported in some studies [3]. Evaluating ammonia levels can also prove helpful during the process of diagnosing argininemia. Most studies support the measurement of ammonia in the context of NBS or in combination with other laboratory tests that detect the absence of red blood cell arginase activity and/or increased plasma arginine. Increased ammonia levels have also been used as triage, followed by genetic or laboratory testing to confirm the diagnosis [3]. Genetic testing--conducted by sequence analysis, deletion/duplication analysis or with a multi-gene panel containing ARG1 gene in tandem with other genes--in order to discover pathogenic mutations in the ARG1 gene is widespread [67,69]. Physical examination, electroencephalogram (EEG), brain imaging, and family history of ARG1-D provide additional diagnostic clues [3]. NBS for arginine--in countries where it is performed--is crucial for early disease detection and prompt initiation of treatment [5]. For instance, on the US Recommended Uniform Screening Panel ARG1-D is incorporated as a secondary target and its diagnosis is established by assessing arginine levels in dried blood spots via tandem mass spectrometry [68]. In cases of known ARG1 gene mutations, prenatal diagnosis is feasible through mutation analysis in chorionic villous tissue, amniocytes, or percutaneous umbilical blood sampling, given that ARG1 is expressed in fetal red blood cells as early as 16-20 weeks of pregnancy at comparable levels to postnatal levels [67,68]. As far as the treatment is concerned, the primary goal in arginineemia is to reduce arginine concentration in plasma below 200 umol/L. Achieving this objective necessitates severe dietary protein restriction, coupled with essential amino acid supplementation, which covers up to 50% of the protein requirement [67]. While hyperammonium occurs less frequently in hyperarginine patients compared to other UCDs, nitrogen scavengers, such as benzoate, phenylbutyrate, and phenylacetate, can be administered to reduce the risk of such crises or treat them when they occur [3,67]. Nitrogen scavengers provide an alternative pathway for waste nitrogen excretion by facilitating the formation and excretion of hippuric acid and phenylacetylglutamine [70]. Ornithine supplementation proves useful by replenishing hepatocellular ornithine--therefore preventing hyperammonium--and simultaneously by inhibiting the formation of neurotoxic guanidino compounds through blockage of AGAT [71]. Lysine supplementation can possibly increase argininuria and can also compete with arginine for uptake in the brain, potentially lowering brain arginine levels [72,73].