Guanidinoacetate Research Articles

Guanidinoacetic acid provides superior cardioprotection to its combined use with betaine and (or) creatine in HIIT-trained rats.

This study aimed to determine how guanidinoacetic acid (GAA) or its combined administration with betaine (B) or creatine (C) influences the cardiac function, morphometric parameters, and redox status of rats subjected to high-intensity interval training (HIIT). This research was conducted on male Wistar albino rats exposed to HIIT for 4 weeks. The animals were randomly divided into five groups: HIIT, HIIT+GAA, HIIT+GAA+C, HIIT+GAA+B, and HIIT+GAA+C+B. After completing the training protocol, GAA (300mg/kg), C (280mg/kg), and B (300mg/kg) were applied daily per os for 4 weeks. GAA supplementation in combination with HIIT significantly decreased the level of both systemic and cardiac prooxidants ( , H2O2, , and thiobarbituric acid reactive substances) compared with nontreated HIIT (p<0.05). Also, GAA treatment led to an increase in glutathione and superoxide dismutase levels. None of the treatment regimens altered cardiac function. A larger degree of cardiomyocyte hypertrophy was observed in the HIIT+GAA group, which was reflected through an increase of the cross-sectional area of 27% (p<0.05) and that of the left ventricle wall thickness of 27% (p<0.05). Since we showed that GAA in combination with HIIT may ameliorate oxidative stress and does not alter cardiac function, the present study is a basis for future research exploring the mechanisms of cardioprotection induced by this supplement in an HIIT scenario.

Dietary guanidinoacetic acid supplementation improves water holding capacity and lowers free amino acid concentration of fresh meat in finishing pigs fed with various dietary protein levels.

The current study was carried out to detect the effect of dietary guanidinoacetic acid (GAA) supplementation on carcass characteristics and meat quality in finishing pigs fed different dietary crude protein (CP) levels. Sixty-four barrows with an initial body weight of 73.05 ± 2.34 kg were randomly allocated into 1 of 4 dietary treatments in a 2 (100% vs. 125% NRC CP level) × 2 (0 vs. 300 mg/kg GAA) factorial arrangement (n = 7). The feeding trial lasted for 49 d. GAA supplementation significantly reduced drip loss (P = 0.01), free water distribution (T23 peak area ratio) (P = 0.05) and the concentrations of free alanine, threonine, methionine and isoleucine (P < 0.05); but increased total glycine content (P = 0.03) in the longissimus dorsi muscle of finishing pigs regardless of the dietary CP levels. Furthermore, primary myogenic cell differentiation system was employed to investigate the influence of inclusion of GAA on free amino acid concentrations in myotubes (n = 4) and validate the finding in the animal feeding trial. We found that GAA inclusion in culture medium also decreased intracellular concentrations of free alanine, threonine, methionine, isoleucine, valine and proline in differentiated primary myogenic cells in vitro (P < 0.05). Meanwhile, relative to diets with 100% NRC CP level, the intake of diets with 125% NRC CP level improved sarcoplasmic protein solubility, increased the contents of carnosine and total free amino acids as well as flavor amino acids in the longissimus dorsi muscle and decreased backfat thickness at the 6–7th ribs in pigs (P < 0.05). In addition, we observed that the impact of dietary GAA supplementation on the last rib fat thickness, shear force, and free lysine content in the longissimus dorsi muscle was dependent on dietary CP levels (P < 0.05). Collectively, dietary GAA supplementation can reduce drip loss, decrease the concentrations of free amino acids and flavor amino acids of fresh meat independent of dietary CP levels.

Skeletal muscle mass, meat quality and antioxidant status in growing lambs supplemented with guanidinoacetic acid

Guanidinoacetic acid (GAA) exists naturally as a precursor of creatine, which possesses several biological functions. In the present study, the effects of dietary GAA supplementation on skeletal muscle mass and meat quality of lambs were investigated. The GAA supplementation increased final body weight, promoted muscle mass and changed the distribution of myofiber size. Meanwhile, elevated ultimate pH and water holding capacity (WHC) of resulting meat were observed in GAA fed lambs. Moreover, the total antioxidative capacity was elevated. Dietary GAA accelerated myofibril protein synthesis through regulation with IGF-1/Akt/mTOR signaling pathway and minimized protein breakdown via regulating abundances of myostatin and phosphorylated FoxO1. In vitro, GAA treatment inhibited sheep primary myoblasts proliferation, and enhanced its myogenic potential. Collectively, these results suggested that GAA might be a feed additive for use by the lamb meat industry as it has potential to improve growth performance, antioxidant status and WHC of resulting meat.

Influence of dietary supplemental guanidinoacetic acid on performance, haematological parameters, carcass characteristics and enzyme activities in male broilers with cold-induced ascites

ABSTRACT This study aimed to evaluate the effects of dietary guanidinoacetic acid (GAA) on performance, enzyme activity and biochemical variables of broilers with cold-induced ascites. A total of 640 day-old male broiler chicks (Cobb 500) were randomly assigned to four treatments (with 8 replicates of 20 birds) including a control and diets supplemented with 0.6, 1.2 and 1.8 g of GAA per kg of feed. On day 14, the temperature was reduced to 17°C during the daytime and 14°C at night. Dietary inclusion of 0.6 g/kg GAA decreased BWG in compared to those fed other levels in the grower period (P < 0.05). The ratio of right to total ventricle, mortality and relative weights of liver were lower in birds fed the diet with 1.2 g/kg GAA compared to bids fed other levels (P < 0.05). The plasma activity of creatine kinase (CK) increased in the birds fed with 1.2 and 1.8 g/kg GAA compared to chicks fed the control diet. It can be concluded that dietary supplemental 1.2 g/kg GAA would be a beneficial way to decrease the mortality rate of broiler reared under cold stress conditions. In addition, the blood levels of HDL-C increased in broilers fed the 1.2 g/kg GAA diet.

The effect of Guanidinoacetic Acid Supplementation on Behavior and Welfare of Broiler Chickens Reared at Two Stocking Densities

The effect of Guanidinoacetic Acid Supplementation on Behavior and Welfare of Broiler Chickens Reared at Two Stocking Densities

Study Protocol: Molecular Imaging of Mouse Brain to Investigate Effect of Pre-made Creatine for Neurodevelopmental Disorders in Neonates

Abstract Objectives The global incidence of preterm births is 11% of all births worldwide each year. With advancement of perinatal care, an increasing number of preterm babies survive. However, this increasing survival does not couple with a reduction in the impaired neurodevelopment. Preterm babies receive parenteral nutrition (PN) in the first few weeks to months of their life. Current PN does not contain creatine. In my previous work, 2-weeks of PN reduced the plasma creatine and its intermediate, guanidinoacetate (GAA) compared to neonatal piglets fed PN with premade creatine. L-arginine: glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) synthesize GAA and creatine. In-vitro activities of AGAT and GAMT in the whole brain of neonates were negligible suggesting neonatal brains may rely on exogenous creatine. Prefrontal cortex (PFC) and hippocampus (Hip) are implicated in cognitive development and memory such as those associated with neurodevelopmental disorders of preterm neonates. We optimized mRNA molecular imaging technique to measure multiple mRNAs in a single brain section. Objective 1: measure distribution pattern and co-expression of AGAT and GAMT enzymes in PFC and Hip. Creatine is a neurotransmitter and an agonist for postsynaptic Gamma-aminobutyric acid (GABA) a receptor. Altered GABAa receptor signalling is implicated in neurodevelopmental disorders. Objective 2: how lack of creatine affects GABAa receptor mRNA expression in PFC and Hip regions of neonatal brain. Methods Methods: Sixteen C57BL6 postpartum dams will be randomly assigned to 2 groups and administer an intraperitoneal injection of 1) creatine (160 mg/kg of body weight) or 2) saline once a week. The blood will be sampled from pups once a week from tail snip to measure circulating GAA and creatine concentrations of pups. Pups will be sacrificed after 4 weeks of nursing and extracted brains will be used for molecular imaging of mRNAs (AGAT, GAMT and GABAa receptor). Results This is proposed study protocol. Conclusions Investigating the co-expression of AGAT and GAMT in brain regions will be novel finding from this proposed protocol. Pre-made creatine will reduce brain AGAT and GAMT mRNA expression but increase GABAa receptor mRNA expression. This will suggest that exogenous creatine is important for Hip and PFC GABAa receptor expression. Funding Sources Not decided.

P27. Effects of adding guanidinoacetic acid to sow feed during gestation and lactation on sow and litter performance

P27. Effects of adding guanidinoacetic acid to sow feed during gestation and lactation on sow and litter performance

Guanidinoacetic Acid as a Nutritional Adjuvant to Multiple Sclerosis Therapy.

Tackling impaired bioenergetics in multiple sclerosis (MS) has been recently recognized as an innovative approach with therapeutic potential. Guanidinoacetic acid (GAA) is an experimental nutrient that plays a significant role in high-energy phosphate metabolism. The preliminary trials suggest beneficial effects of supplemental GAA in MS, with GAA augments biomarkers of brain energy metabolism and improves patient-reported features of the disease. GAA can also impact other metabolic footprints of MS, including demyelination, oxidative stress, and GABA-glutamate imbalance. In this mini-review article, we summarize studies evaluating GAA effectiveness in MS, explore mechanisms of GAA action, and discuss the challenges of using dietary GAA as an element of MS therapy.

Letter to editors: misinterpretation of results led to a biased view about Guanidino acetic acid being beneficial in energy deficient diets

View responses to this article:Response to letter to the editors by Behnam Saremi referring to the article ‘Feeding guanidinoacetic acid to broiler chickens can compensate for low dietary metabolizable energy formulation’ by Pirgozliev et al

Safety and efficacy of a feed additive consisting of guanidinoacetic acid for all animal species (Alzchem Trostberg GmbH).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of guanidinoacetic acid (GAA) when used as nutritional additive in feed and water for drinking for all animal species. The FEEDAP Panel concludes that GAA at 1,200 mg/kg complete feed is safe for chickens for fattening, piglets and pigs for fattening. This concentration in complete feed would correspond to maximum concentrations in water of 600 mg GAA/L for chickens for fattening, piglets and pigs for fattening. The Panel is not in a position to conclude on a safe level of GAA in laying/reproductive birds. In the absence of data on ruminants and salmonids, the FEEDAP Panel cannot conclude on the safety of GAA for all animal species. There is no concern on consumer safety resulting from the use of GAA in feed for poultry and pigs at the proposed conditions of use. The limited data do not allow to conclude on the safety for the consumer when the additive is used in feed for ruminants or fish. GAA is not toxic by inhalation, it is not an irritant to skin and eyes, and it is not a dermal sensitiser. The FEEDAP Panel concludes that the use of GAA as feed additive is not expected to pose a risk to the environment. The use of the additive under assessment in animal nutrition at the proposed conditions of use has the potential to be efficacious in all growing avian, Suidae and ruminant (except for preruminants) species; in growing fin fish other than salmonids and in frog. It is not possible to conclude on the efficacy of the additive in other species, and in reproductive animals.

Supplementation of guanidinoacetic acid and rumen-protected methionine increased growth performance and meat quality of Tan lambs.

ObjectiveTan lambs (n = 36, 3 mo old, 19.1±0.53 kg) were used to assess effects of dietary guanidinoacetic acid (GAA) and rumen-protected methionine (RPM) on growth performance, carcass traits, meat quality, and serum parameters.MethodsLambs were randomly assigned to three treatment groups, with 6 pens per group and 2 lambs per pen. Dietary treatments were: basal diet alone (I); basal diet supplemented with 0.08% GAA+0.06% RPM (II); and basal diet supplemented with 0.08% GAA+0.08% RPM (III). Diets were provided three times a day for 90 d. Intake per pen was recorded daily and individual lamb body weight (BW) was measured monthly. Carcass traits were measured after slaughter and meat quality at the end of the experiment, blood samples were taken on a subgroup of lambs for analysis of indicators mostly related to protein metabolism.ResultsFinal BW and average daily gain for the first and second month, and for the entire experiment were greater in Treatment II compared to Treatment I (p<0.05), whereas feed to gain ratio was lower (p<0.05). Treatment II had the optimal dressing percentage and net meat weight proportion, as well as crude protein and intramuscular fat concentrations in muscles. Treatment II improved meat quality, as indicated by the greater water holding capacity, pH after 45 min and 48 h, and lower shear force and cooking loss. Dietary supplementation of GAA and RPM also increased the meat color a* and b* values at 24 h. Finally, Treatment II increased total protein, and serum concentrations of albumin and creatinine, but decreased serum urea nitrogen concentrations, indicating improved protein efficiency.ConclusionIn this study, 0.08% GAA+0.06% RPM supplementation improved growth performance and meat quality of Tan lambs.

Pilot Study on Acute Effects of Pharmacological Intraperitoneal L-Homoarginine on Homeostasis of Lysine and Other Amino Acids in a Rat Model of Isoprenaline-Induced Takotsubo Cardiomyopathy.

L-Arginine:glycine amidinotransferase (AGAT) catalyzes the formation of L-homoarginine (hArg) and L-ornithine (Orn) from L-arginine (Arg) and L-lysine (Lys): Arg + Lys ↔ hArg + Orn; equilibrium constant KhArg. AGAT also catalyzes the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly ↔ GAA + Orn; equilibrium constant KGAA. In humans, pharmacological hArg is metabolized to Lys. Low circulating and low excretory concentrations of hArg are associated with worse outcomes and mortality in the renal and cardiovascular systems. The metabolism and pharmacology of hArg have been little investigated. In the present study, we investigated the effects of pharmacological hArg (i.p., 0, 20, 220, 440 mg/kg at time point 0 min) on amino acids homeostasis in a rat model of isoprenaline-induced takotsubo cardiomyopathy (i.p., 50 mg/kg at time point 15 min). We measured by gas chromatography-mass spectrometry free and proteinic amino acids, as well as the polyamines putrescine and spermidine in the heart, lung, kidney, and liver of ten rats sacrificed at various time points (range, 0 to 126 min). hArg administration resulted in multiple changes in the tissue contents of several free and proteinic amino acids, as well as in the putrescine-spermidine molar ratio, an indicator of polyamines catabolism. Our results suggest that Lys and Arg are major metabolites of pharmacological hArg. Kidneys and heart seem to play a major metabolic role for hArg. Circulating Lys does not change over time, yet there is a considerable interchange of free Lys between organs, notably kidney and heart, during the presence of isoprenaline in the rats (time range, 15 to 90 min). Antidromic changes were observed for KhArg and KGAA, notably in the heart in this time window. Our study shows for the first time that free hArg and sarcosine (N-methylglycine) are positively associated with each other. The acute effects of high-dosed hArg administration and isoprenaline on various amino acids and on AGAT-catalyzed reaction in the heart, lung, kidney, and liver are detailed and discussed.

Dietary guanidinoacetic acid increases the longissimus dorsi muscle depth of finishing pigs without requiring a higher standardised ileal digestible methionine + cysteine concentration

The objective of this study was to evaluate the interaction of guanidinoacetic acid (GAA) with standardised ileal digestible methionine + cysteine (SID Met + Cys) levels in finishing pig diets on the performance, carcase characteristics, pork quality, blood variables, gene expression of creatine transporter (SLC6A8) and oxidant and antioxidant action. The experimental design (n = 32) was in randomised blocks arranged in a 2 × 2 factorial scheme, consisting of two levels of guanidinoacetic acid (0.00% and 0.05%) and two levels of SID Met + Cys (0.44% and 0.50%). There was no interaction of SID Met + Cys and GAA levels on the performance variables, carcass characteristics, pork quality and creatine transporter gene expression (p ≥ .05), except for plasma lactate dehydrogenase (LDH) (p = .016), wherein at the highest SID Met + Cys level the GAA supplementation (0.05%) showed lower plasma LDH concentration in relation to non-supplementation. Longissimus dorsi muscle depth (MD) (p = .001) showed an isolated effect for the evaluated GAA levels, in which the supplementation increased the MD. Blood urea presented an isolated effect (p = .040) for SID Met + Cys levels, showing lower plasma concentration for the highest dietary level. It was concluded that GAA does not require a higher intake of SID Met + Cys in finishing pig diets and its supplementation improved the depth of Longissimus dorsi muscle, without changing the performance, meat quality and gene expression of the SLC6A8 transporter in the liver. Highlights Higher levels of SID Met + Cys reduced homocysteine concentration, and GAA supplementation reduced plasma lactate dehydrogenase concentration at higher dietary SID Met + Cys level. GAA supplementation improves the depth of Longissimus dorsi muscle and did not affect the post-mortem glycolysis. Higher concentration of SID Met + Cys in finishing pig diets do not affect the gene expression of the creatine transporter (SLC6A8) in the liver.

112 A Meta-Analysis on the Effect of Guanidinoacetic Acid Supplementation on Growth Performance of Growing Pigs

Abstract Guanidinoacetic acid (GAA) is produced in the kidney from arginine and glycine and transported to the liver for the synthesis of creatine. Dietary GAA supplementation can have arginine and energy-sparing effects. The objective of this meta-analysis was to assess the effect size of GAA in growing pigs based on available literature. Using internet search (until October 31, 2021), we identified 7 scientific publications. Only studies that 1) reported growth performance of growing pigs (7 to 107 kg BW), 2) had control without GAA supplementation, and 3) did not aim to demonstrate GAA sparing effects of energy or arginine were selected. From the 7 publications, 4 publications met the criteria, and 2 of the publications contained 2 studies, therefore, a total of 6 studies were included in the meta-analysis. The statistical analysis utilized a random effect model to estimate the mean effect size (MES) of the difference between control and GAA-supplemented pigs, the 95% confidence interval of MES, the probability of MES being different from zero, the probability of the studies to share a common effect size (heterogeneity), and no publication bias. The data were analyzed using SAS 9.4 using a SAS macro created by Rendina-Gobioff and Kromrey (2006). Summary statistics and average change in percentage are reported in Table 1. Pigs fed GAA-supplemented diets had greater (P &amp;lt; 0.001) final BW, ADG, and G:F, and the magnitude of the effect is considered to be large (&amp;gt;|0.8|). GAA did not influence (P = 0.976) ADFI. The presence of heterogeneity was observed in all the responses, suggesting that the effect size varies among studies. Publication bias was not found for any of the responses. Overall, supplementation of GAA increased BW, ADG, and G:F by 1.68, 3.95 and 7.01%, respectively, and the effect size varies, potentially due to the inclusion rate and duration of supplementation.

Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities

Creatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism.

Managing broiler production challenges at high altitude.

This review covers the challenges of broiler chickens at high altitude, with the focus on growth performance and physiological response. The review also sheds light on nutritional and management interventions that help overcome the challenges raised at high altitude. Reduced concentration of atmospheric oxygen is by far the biggest challenge that remarkably affect growth performance and livability of broiler chickens reared in high altitude area. Broiler chickens have endured intensive genetic selection, which potentially predispose them to several metabolic disorders. Hypoxia is an overriding factor that may increase the incidence of metabolic disorders, mainly ascites syndrome at high altitude. Commercial broiler strains cannot fully achieve their genetic potential when raising at highland regions. Careful nutrition and management considerations are required to prevent metabolic disorders when raising broilers at high altitude. In ovo or in‐feed nutraceuticals such as l‐carnitine and guanidinoacetic acid as well as pharmaceuticals, texture of feed and the use of proper sources and levels of dietary energy and protein are important factors that need to be carefully considered for rearing broiler chickens at high altitude. Management strategies such as lighting programs have been shown to be effective to circumvent ascites prevalence. Special breeding programs may also be considered to develop strains with resistance to ascites.

Effects of dietary guanidinoacetic acid on growth performance, guanidinoacetic acid absorption and creatine metabolism of lambs.

Guanidinoacetic acid (GAA) is the only precursor for the creatine synthesis of vertebrates. Creatine (Cr) and phosphocreatine (PCr) are able to provide energy for the rapid growth and development of the muscle tissue. This study evaluated the effects of dietary different levels GAA on growth performance, GAA absorption and creatine metabolism of lambs. Twenty-four 3-month-old healthy Kazakh male lambs (body weight = 27.35± 0.58 kg) were randomly divided into four groups with 6 lambs in each group. The lambs were fed with the basal diets supplemented with 0 (0 mg/kg group), 500 (500 mg/kg group), 1000 (1000 mg/kg group) and 1500 mg (1500 mg/kg group) GAA per kg diet (DM basis), respectively. The results showed that, as the GAA content of the diet increased, there was a quadratic change in DMI, with the lowest in the 500 mg/kg group and the highest in the 0 mg/kg group. The CK enzyme activity and ATP content in quadriceps muscle increased linearly with increasing levels of diary GAA in the diet. PCr levels and ADP levels in the longest dorsal muscle increased linearly with increasing levels of GAA in the diet. The relative expression of SLC6A6 and SLC6A8 mRNA in the jejunum and ileum mucosa showed a quadratic change as the dietary GAA level increased, with the lowest relative expression in both the 1500 mg/kg group. With the increase of dietary GAA level, both Cr concentration in hepatic vein plasma and the portal plasma GAA concentration shows a quadratic change, with the highest concentration in the 500 mg/kg group and the lowest concentration in the 0 mg/kg group. Therefore, dietary supplementation with 500~1000 mg/kg DM GAA is recommended for lambs.

Biosynthesis of Guanidinoacetate by Bacillus subtilis Whole-Cell Catalysis

Guanidinoacetate (GAA) is a naturally occurring amino acid derivative and the direct precursor of creatine, which is widely used in feed additives and the pharmaceutical industry. The current industrial synthesis of GAA is based on chemical methods, which limits the application of GAA. Here, a biological approach is developed for food safety GAA production via whole-cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis. First, we introduced a heterologous arginine: glycine amidinotransferase (AgaT) from Amycolatopsis kentuckyensis into B. subtilis and optimized its expression level using strategies including: promoter optimization, ribosome binding site (RBS) and N-terminal coding sequence (NCS) screening. In order to alleviate the waste of arginine and the inhibition of AgaT by ornithine, we optimized the natural ornithine cycle in B. subtilis. At the same time, the first gene in the glycine degradation pathway was knocked out. After optimization using these strategies, the titer of GAA was 4.26 g/L with a productivity of 0.21 g/L/h in 20 h, which provides a new method for the biosynthesis of GAA.

Method modification to reduce false positives for newborn screening of guanidinoacetate methyltransferase deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder that results in reduced activity of guanidinoacetate methyltransferase, an accumulation of guanidinoacetate (GUAC), and a lack of cerebral creatine (CRE). Lack of CRE in the brain can cause intellectual disability, autistic-like behavior, seizures, and movement disorders. Identification at birth and immediate therapy can prevent intellectual disability and seizures. If started early in life, treatment with creatine supplements is highly effective. Because there are reliable biomarkers for GAMT deficiency, GUAC and CRE, and because the disorder is readily treatable with a significant improvement in outcomes, GAMT deficiency is an excellent candidate for newborn screening. Several programs have conducted pilot programs or started screening. An isobaric interferant of the GUAC marker has been reported which may cause false positive results. To reduce the number of false positives, a second-tier HPLC test to separate GUAC from unknown, isobaric interferants may be incorporated into the screening algorithm. New York State began screening for GAMT deficiency in October 2018 using a three-tiered screening approach. Quantification of GUAC and CRE were incorporated into routine screening for amino acids and acylcarnitines. In the first year of screening a total of 263,739 samples were tested for GAMT deficiency. Of these, 3382 required second tier testing. After second tier testing, 210 repeat specimens were requested for borderline results and 10 referrals were made to specialty care centers for confirmatory testing. In the first year of screening there were no confirmed cases of GAMT deficiency detected. To reduce the number of samples needing second tier testing and the number false positives we explored the use of a second MS transition to confirm the identity of the GUAC marker. GUAC and its internal standard are detected as butylated esters after sample preparation and derivatization. The original method used transition of the GUAC molecular ion of m/z 174.1 to a reactant ion of m/z 101.1. To confirm the identity of the GUAC marker we selected a qualifier ion of 174.1 > 73. The alternative product ion results were found to agree more closely with the second tier HPLC-MS/MS results for GUAC. It was found that the alternative transition may be used for quantification of the GUAC marker with acceptable analytical performance (linearity, accuracy, and precision). On March 5, 2020, the method of analysis for GUAC was modified to use the alternative product ion. For a comparable 6-month period, the modified method reduced the number of samples requiring second tier testing by 98%, reduced the number of borderline results requiring a repeat sample by 87.5%, and reduced the number of referrals to specialty care centers by 85%. Using the modified method, the correlation (r-squared) of the first and second tier screening results for GUAC is greater than 0.95. Since the first-tier results correlate well with the second-tier results, the second-tier screening is no longer necessary with the modified method.

Effects of Creatine Supplementation on Brain Function and Health.

While the vast majority of research involving creatine supplementation has focused on skeletal muscle, there is a small body of accumulating research that has focused on creatine and the brain. Preliminary studies indicate that creatine supplementation (and guanidinoacetic acid; GAA) has the ability to increase brain creatine content in humans. Furthermore, creatine has shown some promise for attenuating symptoms of concussion, mild traumatic brain injury and depression but its effect on neurodegenerative diseases appears to be lacking. The purpose of this narrative review is to summarize the current body of research pertaining to creatine supplementation on total creatine and phophorylcreatine (PCr) content, explore GAA as an alternative or adjunct to creatine supplementation on brain creatine uptake, assess the impact of creatine on cognition with a focus on sleep deprivation, discuss the effects of creatine supplementation on a variety of neurological and mental health conditions, and outline recent advances on creatine supplementation as a neuroprotective supplement following traumatic brain injury or concussion.