Changes in BCAA levels in Parkinson’s disease

BCAAs are essential amino acids that are required for growth, development, nutrient signaling, providing nitrogen to neurotransmitter synthesis and glutamate/glutamine cycling. These are crucial roles of BCAAs to maintain body and brain health. However, in recent years, there is an important debate about BCAA’s effects on neurodegenerative diseases that has arisen.

While some research groups suggest that BCAAs may benefit brain function and cognitive aging, there are differing hypotheses about their role in neurodegenerative diseases. ^{Reference Coppola, Wenner and Ilkayeva35–Reference Yan, Yang and Sun41} One hypothesis is that excessive dietary intake of BCAAs may interfere with the uptake of neutral amino acids such as tryptophan and tyrosine, which are essential for serotonin and dopamine production. BCAAs and these neutral amino acids share the same transporters across the blood-brain barrier. Therefore, high BCAA levels could compete with tryptophan and tyrosine for transport into the brain, potentially impairing the synthesis of 5-hydroxytryptophan (5-HT) and dopamine. Our cumulative results showed a significant increase in valine levels in the PD group, while levels of isoleucine and leucine did not change significantly. This suggests that the altered BCAA profile, particularly the increase in valine, may influence the balance of neurotransmitter synthesis, potentially contributing to the neurochemical disruptions observed in PD.

A study reported that high plasma BCAA levels resulted in low central 5-HT levels. Anxiety-like behavioral changes were observed in rats fed a high-fat, high-carbohydrate diet enriched with BCAAs. ^{Reference Coppola, Wenner and Ilkayeva35} With BCAA supplementation, a decrease was observed in tryptophan and its metabolite kynurenic acid. ^{Reference Coppola, Wenner and Ilkayeva35} Oral BCAA supplementation during exercise has been shown to reduce brain catecholamine levels. ^{Reference Choi, DiSilvio, Fernstrom and Fernstrom36} In another study, it was determined that tyrosine and dopamine levels in the brain were decreased with BCAA supplementation. ^{Reference Le Masurier, Oldenzeil, Lehman, Cowen and Sharp37} In a study conducted on healthy people, when they evaluated the effect of BCAA supplementation on dopamine levels by plasma prolactin levels, it was shown that BCAA supplementation could cause an increase in prolactin secretion by suppressing dopamine levels. ^{Reference Neuhaus, Goldberg and Hassoun38} In addition, the BCAA supplements used in the studies typically show short-term effects, and it remains unclear how neurotransmitter levels might be affected by long-term BCAA consumption over extended periods. ^{Reference Yoo, Shanmugalingam and Smith39}

The study by Zhang et al. (2022), which is also included in this meta-analysis, found that patients with PD have lower plasma BCAA and aromatic amino acid levels. ^{Reference Zhang, He and Qian15} Another study investigating the effects of whey protein supplementation, which is a good source of BCAAs, on PD showed favorable results. ^{Reference Yoo, Shanmugalingam and Smith39} With the whey protein intake, the serum homocysteine level was decreased, while oxidized glutathione level was increased in PD patients. However, they also reported that there were no changes in the clinical outcomes. ^{Reference Tosukhowong, Boonla and Dissayabutra40} Also, in a recent animal study, researchers revealed that rotenone-induced PD may lead to alteration in the gut microbiota, which may be the possible cause of peripheral BCAA deficiency. When they fed the animals with a high BCAAs diet, they found improvements in the inflammatory levels, motor and non-motor dysfunctions and dopaminergic neuron impairment in PD mice. ^{Reference Yan, Yang and Sun41}

A very recent study in 2023, which analyzed data from 21,982 Alzheimer’s disease (AD) cases and 41,944 controls, found an association between decreased levels of BCAAs and the presence of AD in patients. ^{Reference Qian, Liu and Zhang42}

A study with app/ps1 double transgenic mice published in the Cells journal in 2022 reports that higher plasma BCAA levels were displayed in this AD model, and reducing BCAA daily intake alleviates AD-related pathology and cognitive decline. The same study highlighted a potential link between BCAAs and AD progression. ^{Reference Siddik, Mullins and Kramer43}

Furthermore, the consumption of BCAAs in a high-fat diet may be an important factor for neurological impairment as shown in an animal study of Alzheimer’s disease model. Mice fed with a high BCAA-high-fat diet showed higher tau neuropathology, and only 4 out of 13 animals survived. Mice fed with a low-BCAA diet showed higher threonine and tryptophan cortical levels. ^{Reference Tournissac, Vandal and Tremblay44}

In a recent study in 2023, leucine-rich α2 glycoprotein was investigated to be a biomarker of systemic inflammation in PD in 66 patients and 31 age-matched controls. It was significantly higher in the patient group than in the control group and also correlated with Charlson comorbidity index, C-reactive protein levels and dementia. ^{Reference Ohmichi, Kasai and Shinomoto45}

There are conflicting results in the literature; however, our study may help to clarify the relationship between BCAA intake and PD and neurological functions. By examining the serum levels of BCAAs in PD patients, as well as the mechanistic relationships of transporters, the effects of BCAA supplements and their potential impact on disease progression in the literature, our findings provide a more comprehensive understanding of how BCAAs influence PD and may inform future research and therapeutic strategies. It might be better to separate BCAAs as each of these amino acids might be changed in patients or affect the disease progression in a different way. To understand the underlying mechanism, more studies on BCAA’s effects on brain and neurodegenerative disease development are needed. Also, it is important to examine the BCAAs in the other dietary and lifestyle factors that can change the effects of amino acids and disease progression.

Altered proline metabolism

Besides BCAAs, there were several amino acids found to be significantly changed in the patients compared to control.

Our results showed that proline levels were significantly higher in the PD group than in the controls; however, alanine and glycine levels weren’t changed.

Proline constitutes one-third of amino acids in the collagen proteins, comprising nearly 30% of body proteins. To understand the increase in proline levels it is beneficial to examine the stress response of the body. L-proline has the role of a chemical chaperone, preventing protein unfolding or misfolding for endoplasmic reticulum stress; ^{Reference Liang, Dickman and Becker46} however, at high levels, it can be harmful to neural activity.

L-proline metabolism might be related to the development of neurodegenerative diseases associated with the formation of protein aggregates, such as Parkinson’s, Alzheimer’s, etc. These diseases have a number of common features in their processes. Energy metabolism dysfunction, glutamate excitotoxicity and oxidative stress seem to be underlying in the pathophysiology of these disorders. ^{Reference Nagaoka, Kunii and Hino47}

The process of proline catabolism involves the conversion of proline to pyrroline-5-carboxylate (P5C) by proline dehydrogenase (PRODH) in the mitochondrial matrix. This generates glutamate through NAD-dependent P5C dehydrogenase (P5CDH), which is an essential excitatory neurotransmitter in neurons and a precursor to glutamine, GABA and mitochondrial TCA cycle intermediates. On the other hand, glutamate can be converted into a P5C intermediate through P5C synthase (P5 CS) and further reduced to proline by P5C reductases (PYCRs). All the enzymes involved in the proline metabolism pathway have been reported to be associated with neurological or psychiatric disorders in human and animal models. ^{Reference Guo, Tang, Yang and Li48–Reference Liu, Borchert and Donald50} Also, hypovitaminosis B complex especially vitamin B₆ and B₁₂ are important for proline metabolism and hyperhomocysteinemia through impaired remethylation.

As a part of neural metabolism, proline has the capability to function as a metabolic precursor of L-glutamate. PRODH knockout mice have shown that the cytosolic accumulation of L-proline disrupts GABAergic transmission through glutamate decarboxylase blockade. ^{Reference Cohen and Nadler51} Moreover, normally, proline induces L-glutamate synthesis and acts as a GABA-mimetic inhibitor of the GAD enzyme. ^{Reference Crabtree, Park, Gordon and Gogos52} Therefore, proline can reduce the synthesis of the GABA neurotransmitter and accumulation of L-glutamate, thereby leading to synaptic dysfunction in some forms of disease. ^{Reference Patriarca, Cermola and D’Aniello53} On the other hand, if proline cannot contribute glutamate or collagen synthesis due to the lack of these metabolic paths, it may accumulate. Dysfunctional proline metabolism could be one of the underlying mechanisms of muscle weakness in PD. Further studies are needed to elucidate this hypothesis.

Related to this excitotoxicity, particularly in neural glutamate metabolism, patients with genetic defects in PRODH or in P5CDH, called hyperprolinemia I/hyperprolinemia II, have higher proline levels up to 10–15-fold than in normal people and may have developed schizophrenia-related phenotypes (learning, memory and sensorimotor gating) and schizophrenia. ^{Reference Nagaoka, Kunii and Hino47,Reference Clelland, Read and Baraldi54}

Studies have shown that stress and anxiety can influence the concentrations of hydroxyproline and proline in urine. ^{Reference Lee, Kim, Park and Lee55} Long-term exposure to proline has also been linked to behavioral changes in zebrafish, which were reversed by antipsychotics, indicating a relationship between proline and psychiatric diseases. ^{Reference Savio, Vuaden, Piato, Bonan and Wyse56} Given these findings, it is important to explore how elevated proline levels observed in PD patients might be related to similar stress or behavioral mechanisms.

In vitro studies have revealed that differentiated neurons depleted of PYCR2 showed thinner neuronal fibers and significantly increased axonal beading, an early morphological hallmark of neuronal injury. ^{Reference Escande-Beillard, Loh and Saleem57} Furthermore, there is a hypothesis that PRODH deficiency could lead to hyperactivation of the dopaminergic system due to dysregulated astroglial control of dopamine homeostasis. ^{Reference de Oliveira Figueiredo, Bondiolotti, Laugeray and Bezzi58} Other studies suggest that in the hypothalamus, proline is taken up by astrocytes and converted into lactate, which is then released from astrocytes and taken up by neurons. In neurons, lactate is oxidized for energy production. ^{Reference Arrieta-Cruz and Gutiérrez-Juárez59}

There is significant evidence that PRODH polymorphisms are associated with susceptibility to schizophrenia and defects in PRODH have been linked to glutamatergic and GABAergic neuron dysfunctions. ^{Reference Guo, Tang, Yang and Li48,Reference Crabtree, Park, Gordon and Gogos52}

In the literature, it was also stated that proline-rich protein 14, which is involved in the alteration and activation of the mTOR signaling pathway, was upregulated in PD patients. ^{Reference Jin, Tan and Shi60} Its expression was found higher in whole blood, substantia nigra and medial substantia in the PD group than in healthy controls. They mentioned that the detection of proline-rich protein 14 levels in serum/plasma can be a biomarker for PD. ^{Reference Jin, Tan and Shi60}

Another reason for proline accumulation might be also related to the arginine-ornithine cycle. Our results showed that while arginine levels in PD patients were decreased, there was a significant increase in ornithine levels. In the urea cycle, ornithine-arginine transferase converts L-ornithine to proline-5 carboxylase, which is a precursor of L-proline via proline-5 carboxylase reductase. ^{Reference Morris61}

Examining proline metabolism could provide valuable insights into the mechanisms underlying neurodegenerative diseases. By understanding how proline levels and their metabolic pathways interact with disease processes, we can gain a better understanding of the causes of PD and potentially identify new therapeutic targets.

Excessive ornithine levels in Parkinson’s disease

Ornithine levels were found to be elevated in PD patients and were also associated with the severity of the disease. Advanced-stage PD patients had higher ornithine levels than early-stage PD patients. Ornithine levels were found correlated with α-synuclein protein, which includes polyamines leading to the accumulation and fibril formation of putrescine, spermine and spermidine. Its accumulation in neurons is playing a key role in PD development. ^{Reference Chang, Cheng, Tang, Huang, Wu and Chen6,Reference Ghosh, Mehra, Sahay, Singh and Maji7}

Polyamines are molecules including two to four amino groups. They play a critical role in several mechanisms in living organisms including apoptosis, cell division and differentiation, cell proliferation, DNA and protein synthesis, gene expression, homeostasis and signal transduction. Polyamine biosynthesis initiates from arginine and ornithine. Arginine is first converted to ornithine, and ornithine decarboxylase leads to putrescine synthesis. Then putrescine can be converted to spermidine and spermine. The accumulation of polyamines has been reported to be related to several diseases. ^{Reference Handa, Fatima and Mattoo8}

The major metabolic ways that can be activated when ornithine levels are increased include the synthasis of L-proline, L-glutamate, GABA and polyamines and the activation of urea cycle. ^{Reference Majumdar, Barchi and Turlapati62} Also, the activation of the urea cycle may contribute to the excessive urea production, which can increase of osmolarity in the cerebellum, cerebral cortex and brain stem. ^{Reference Çelik, Erşan, Kılıçgün, Kapancık and Erşan63} Ornithine-related osmotic pressure also affects plasticity in the hippocampal region with the tonic inhibition of the GABA receptors. ^{Reference Glykys, Mann and Mody64} An in vitro study showed that osmotic stress is also a causative factor for α-synuclein accumulation. ^{Reference Fragniere, Stott and Fazal65}

Homocysteine as a biomarker of pathogenesis

The non-proteinogenic α-amino acid homocysteine is directly linked to diseases such as dementia, heart disease and stroke, with elevated blood levels indicating underlying physiological issues. ^{Reference Smith, Refsum and Bottiglieri66,Reference Kaplan, Tatarkova, Sivonova, Racay and Lehotsky67}

As it is expected and well-studied and approved with meta-analysis studies, homocysteine level was found significantly increased in PD, as high homocysteine leads to nerve cell apoptosis, oxidative stress and DNA damage of neuron cells. ^{Reference Fan, Zhang and Li16,Reference Dong and Wu68} Increased homocysteine levels in PD patients showed an association with frontal cortical thinning and microstructural damage in frontal and posterior-cortical regions. ^{Reference Sampedro, Martínez-Horta and Horta-Barba69}

Therefore, our results suggest that its positive correlation with glutamate and ornithine may need to be considered in the potential relationships that could be established with PD.

Histidine tendency to increase in Parkinson’s disease

We found histidine levels tend to be increased in PD patients, and further sensitivity analysis showed that this was due to one study that gave contrary results to the rest of the studies. ^{Reference Babu, Gupta and Paliwal21} With the elimination of that study, histidine levels significantly increased in the PD group (p: 0.04). The dimerization of α-synuclein protein requires histidine, which is an important aggregation for PD development. ^{Reference Abeyawardhane, Fernández and Heitger70} Histidine is converted into histamine by the enzyme histidine decarboxylase. Regarding the effects of histamine on PD, animal experiments showed that increased histamine levels may lead to the degeneration of dopaminergic neurons in the substantia nigra. ^{Reference Liu, Chen, Liu, Hu and Luo71} The enzyme responsible for the clearance of histamine, histamine N-methyltransferase, has been found to play an important role in the pathogenesis of PD. ^{Reference Shan, Bossers and Luchetti72} A negative correlation was observed between the mRNA expression of histamine N-methyltransferase in the substantia nigra and disease progression inPD patients. ^{Reference Shan, Bossers and Luchetti72} Evidence indicated that excessive histamine production may be related to PD and other neurodegenerative diseases, which are also related to increased histidine amino acid levels. ^{Reference Shan, Bao and Swaab73}

Also, histidine is a precursor of carnosine (β-alanyl-L-histidine), which is a dipeptide synthesized from b-alanine and histidine. Blockage of the carnosine pathway or over-hydrolysis of carnosine may result in histidine accumulation. Carnosine is a neuroprotective agent and a potential additive for Parkinson’s patients. ^{Reference Schön, Mousa and Berk74}

Decrease in specific amino acids in Parkinson’s disease

Motor and non-motor symptoms

Our cumulative analysis revealed correlations between amino acid levels and disease severity indicators such as the UPDRS III score, Hoehn and Yahr stage and disease duration. Notably, arginine levels negatively correlated with the LEDD, suggesting that alterations in arginine metabolism might be associated with the progression of motor symptoms and medication requirements. Arginine serves as a precursor for nitric oxide (NO) synthesis, which is crucial for neurotransmission and neurovascular function. ^{Reference Yabuki, Shioda and Yamamoto80} Elevated levels of neuronal and inducible NO synthase (NOS) have been observed in the substantia nigra of PD patients and animal models. ^{Reference Aquilano, Baldelli, Rotilio and Ciriolo90} The negative correlation between LEDD and decreasing arginine levels may indicate the depletion of arginine for NO synthesis. However, arginine levels were not significantly different between groups, highlighting the need for further studies to clarify its role in PD symptoms.

Similarly, phenylalanine levels negatively correlated with UPDRS III scores, suggesting a potential link between this amino acid and motor symptom severity, although phenylalanine levels were not significantly different between PD and healthy groups. As a precursor to tyrosine, which is then converted to dopamine, lower phenylalanine levels may reduce the availability of tyrosine and subsequently dopamine, ^{Reference Shnitko, Taylor and Stringfield91} potentially leading to more severe motor symptoms as reflected by the UPDRS III score.

Additionally, citrulline levels tended to decrease with disease duration, highlighting its potential role in disease progression. Our findings also showed that citrulline levels were significantly decreased in the PD group compared to controls, indicating a strong mechanism underlying PD. Citrulline, like arginine, is crucial in the NO production pathway. Although NO has neuroprotective effects, ^{Reference Yabuki, Shioda and Yamamoto80} elevated levels of neuronal and inducible NOS observed in the substantia nigra of PD patients ^{Reference Aquilano, Baldelli, Rotilio and Ciriolo90} suggest that disruptions in these pathways could negatively affect neuronal health and function, potentially influencing disease progression.

We acknowledge several limitations in our study that arise from the quality of the studies and the data available in the literature. A primary limitation is the insufficient reporting of amino acid levels by disease stage, gender and ethnicity, which restricted our ability to perform analyses across these variables. Consequently, we were unable to conduct subgroup analyses based on disease stage or demographic characteristics, limiting our exploration of potential variations in amino acid profiles. Additionally, the studies included in our meta-analysis did not provide information on amino acid levels at various stages of PD or before disease onset, precluding an assessment of when these changes first occurred.

Our meta-analysis also encountered challenges due to high statistical heterogeneity, as reflected by the I² values in each amino acid analysis. This heterogeneity is indicative of variations in study design, diagnostic criteria and measurement methods, which can affect the consistency of findings across studies. While clinical heterogeneity is expected in meta-analyses, it underscores the necessity for future research to address these issues by providing more detailed and consistent reporting. Future studies should aim to include data stratified by disease stage and patient characteristics.