Isovalerylglycine
Background
Section titled “Background”Isovalerylglycine is a urinary metabolite that serves as a crucial biomarker in human metabolism. It is a conjugate of isovaleric acid and glycine, formed as part of a detoxification pathway for excess isovaleric acid. The presence and concentration of isovalerylglycine in biological samples, particularly urine, can indicate specific underlying metabolic conditions.
Biological Basis
Section titled “Biological Basis”The formation of isovalerylglycine is directly linked to the metabolism of leucine, an essential branched-chain amino acid. During the catabolism of leucine, an intermediate compound called isovaleryl-CoA is produced. Normally, isovaleryl-CoA is further metabolized by the enzyme isovaleryl-CoA dehydrogenase, encoded by theIVDgene. In individuals with a deficiency or dysfunction of this enzyme, isovaleryl-CoA accumulates. To mitigate the toxic effects of this accumulation, the body conjugates isovaleryl-CoA with glycine, forming isovalerylglycine, which is then excreted in the urine. This metabolic pathway represents an alternative route for the removal of toxic isovaleric acid derivatives.
Clinical Relevance
Section titled “Clinical Relevance”The primary clinical significance of isovalerylglycine lies in its role as a diagnostic marker for Isovaleric Acidemia (IVA), an inherited metabolic disorder. IVA is an autosomal recessive condition caused by mutations in theIVDgene, leading to a deficiency of isovaleryl-CoA dehydrogenase. Elevated levels of isovalerylglycine in urine, alongside other characteristic metabolites, are a key indicator of IVA. Early detection of IVA through newborn screening, often utilizing tandem mass spectrometry to identify elevated isovalerylglycine or its precursor isovalerylcarnitine, is critical for preventing severe neurological damage, developmental delays, and life-threatening metabolic crises associated with the condition.
Social Importance
Section titled “Social Importance”The ability to detect isovalerylglycine provides a vital tool for public health initiatives, particularly newborn screening programs. Early diagnosis of Isovaleric Acidemia allows for prompt initiation of dietary management, supplementation, and other therapeutic interventions, which can significantly improve the long-term prognosis and quality of life for affected individuals. Without early intervention, IVA can lead to severe and irreversible health consequences. The identification of isovalerylglycine as a reliable biomarker has thus had a profound social impact by enabling preventative care and better outcomes for a rare but serious genetic disorder, reducing the burden on families and healthcare systems.
Signs and Symptoms
Section titled “Signs and Symptoms”Clinical Manifestations and Presentation Patterns
Section titled “Clinical Manifestations and Presentation Patterns”Elevated levels of isovalerylglycine are often associated with a spectrum of clinical presentations, ranging from severe neonatal onset to milder, chronic forms. Affected individuals may exhibit poor feeding, vomiting, lethargy, and a distinctive “sweaty feet” odor, which is a key diagnostic clue. In acute metabolic crises, symptoms can progress rapidly to encephalopathy, seizures, and coma, often triggered by increased protein intake, fasting, or illness. The severity of these manifestations can vary significantly, with some individuals experiencing profound neurological impairment and others showing more subtle developmental delays or intermittent episodes of decompensation.
Diagnostic Biomarkers and Measurement Approaches
Section titled “Diagnostic Biomarkers and Measurement Approaches”The presence and elevated concentration of isovalerylglycine serve as a crucial diagnostic biomarker. It is typically measured in urine using analytical techniques such as gas chromatography-mass spectrometry (GC-MS), which provides a definitive identification and quantification of the compound. While isovalerylglycine itself is a direct objective measure, its levels correlate with the severity of clinical signs and symptoms. In newborn screening programs, an elevated C5-acylcarnitine (isovalerylcarnitine) in blood spots often prompts further confirmatory testing, which includes the measurement of isovalerylglycine in urine.
Phenotypic Variability and Heterogeneity
Section titled “Phenotypic Variability and Heterogeneity”The clinical expression associated with isovalerylglycine elevation demonstrates considerable variability, influenced by factors such as residual enzyme activity, genetic background, and environmental triggers. Individuals may present at different ages, from the neonatal period with acute life-threatening crises to later childhood or adulthood with more subtle, chronic, or intermittent symptoms. Age-related changes can lead to a shift in presentation, with some patients developing neurological complications or growth retardation over time. While sex differences are not typically a primary determinant of presentation, individual metabolic demands and dietary habits can influence the frequency and severity of symptomatic episodes.
Diagnostic Significance and Prognostic Indicators
Section titled “Diagnostic Significance and Prognostic Indicators”The detection of elevated isovalerylglycine has high diagnostic value, serving as a definitive marker for a specific organic acidemia. Its presence helps in differentiating this condition from other metabolic disorders that may present with similar non-specific symptoms, such as metabolic acidosis or neurological dysfunction. Early identification through newborn screening and subsequent confirmatory testing for isovalerylglycine is critical, as timely dietary intervention and medical management can significantly improve long-term outcomes and prevent irreversible neurological damage. Persistent or highly elevated levels, especially in untreated individuals, can indicate a more severe prognosis, highlighting the importance of ongoing monitoring and therapeutic adherence.
Causes
Section titled “Causes”The presence and levels of isovalerylglycine, a marker metabolite, are primarily influenced by a complex interplay of genetic predispositions, dietary factors, environmental exposures, and physiological states. Understanding these causal factors is crucial for comprehending its metabolic significance and potential implications for health.
Genetic Basis and Inherited Metabolic Defects
Section titled “Genetic Basis and Inherited Metabolic Defects”The primary cause for elevated isovalerylglycine levels often lies in inherited genetic defects that impair the normal metabolism of the branched-chain amino acid leucine. A classic example is Isovaleric Acidemia (IVA), an autosomal recessive disorder caused by mutations in theIVDgene, which encodes isovaleryl-CoA dehydrogenase. This enzyme is critical for the third step in leucine degradation, converting isovaleryl-CoA to 3-methylcrotonyl-CoA.[1]A deficiency in isovaleryl-CoA dehydrogenase leads to the accumulation of isovaleryl-CoA and its derivatives, including isovaleric acid, which is then conjugated with glycine to form isovalerylglycine for excretion. The specific variants within theIVDgene, such as certain missense or null mutations, dictate the severity of the enzyme deficiency and, consequently, the clinical presentation and the extent of isovalerylglycine accumulation.[2]
Beyond single-gene Mendelian disorders, polygenic factors and gene-gene interactions may also subtly influence isovalerylglycine levels, even in individuals without a frank metabolic disorder. Variations in genes encoding other enzymes in the leucine degradation pathway, or those involved in glycine conjugation or renal excretion, could modulate the overall metabolic flux and the efficiency of isovalerylglycine elimination. While not typically leading to overt clinical disease, such genetic variants might contribute to individual differences in baseline levels or responsiveness to dietary and environmental challenges.
Metabolic Load, Dietary Factors, and Environmental Triggers
Section titled “Metabolic Load, Dietary Factors, and Environmental Triggers”Dietary intake plays a significant role in modulating isovalerylglycine levels, particularly in individuals with an underlying genetic predisposition or a compromised metabolic capacity. A diet rich in protein, especially those high in leucine, directly increases the substrate load for the leucine degradation pathway. In individuals with partialIVDenzyme deficiency or other subtle metabolic impairments, this increased load can overwhelm the remaining enzymatic activity, leading to a rise in isovalerylglycine production and excretion.[3]Conversely, dietary interventions involving leucine restriction are a cornerstone of managing conditions like IVA, demonstrating the direct link between diet and metabolite levels.
Environmental factors, including exposure to certain toxins or medications, can also impact the metabolic pathways that handle leucine and its derivatives. Some drugs might interfere with enzyme function, alter mitochondrial activity, or compete for cofactors, thereby exacerbating an existing metabolic bottleneck. Furthermore, physiological stressors such as infections, fever, or prolonged fasting can trigger metabolic decompensation in susceptible individuals. These stressors increase protein catabolism, releasing more leucine into the system, which, coupled with reduced metabolic efficiency during illness, can lead to a rapid and significant increase in toxic metabolites like isovaleric acid and its glycine conjugate, isovalerylglycine.[4]
Developmental Trajectories and Epigenetic Regulation
Section titled “Developmental Trajectories and Epigenetic Regulation”Early life influences and developmental stages can have a profound impact on the metabolic pathways that govern isovalerylglycine levels. The neonatal period, with its rapid growth and unique nutritional demands, represents a critical window where metabolic vulnerabilities can manifest. For instance, in infants with IVA, the introduction of protein-rich milk after birth can quickly lead to the accumulation of toxic metabolites due to their inability to process leucine effectively.[5]The maturation of metabolic enzymes and pathways throughout childhood and adolescence can also influence how efficiently isovalerylglycine is handled.
Epigenetic mechanisms, such as DNA methylation and histone modifications, may also play a role in regulating the expression of genes involved in leucine metabolism, includingIVD. While direct evidence linking specific epigenetic marks to isovalerylglycine levels is still an area of active research, it is plausible that early life nutritional status, maternal diet during pregnancy, or exposure to environmental factors could induce lasting epigenetic changes. These modifications might subtly alter the expression levels of key metabolic enzymes, influencing an individual’s long-term metabolic capacity and their susceptibility to accumulating metabolites like isovalerylglycine under various conditions.
Interacting Factors: Gene-Environment, Comorbidities, and Age
Section titled “Interacting Factors: Gene-Environment, Comorbidities, and Age”The manifestation of elevated isovalerylglycine levels often results from complex gene-environment interactions. Individuals carrying specific genetic variants that confer a partial deficiency in leucine metabolism might remain asymptomatic under normal dietary and environmental conditions. However, when challenged by a high-protein diet, severe illness, or specific medication, their latent metabolic impairment becomes evident, leading to a significant increase in isovalerylglycine. This highlights how genetic predisposition acts as a susceptibility factor, requiring an environmental trigger to manifest the metabolic phenotype.[6]
Furthermore, the presence of comorbidities can significantly influence isovalerylglycine levels. Conditions affecting liver function, such as hepatic insufficiency, can impair the detoxification and conjugation pathways necessary for isovalerylglycine formation and excretion. Kidney disease can reduce the efficiency of its renal clearance, leading to its accumulation in the body. Certain medications, beyond those directly interfering with enzyme activity, might also impact overall metabolic health or organ function, indirectly contributing to altered isovalerylglycine levels. Finally, age-related changes in metabolic efficiency, organ function, and dietary habits can also play a role, with both very young infants and elderly individuals potentially having reduced capacity to handle metabolic loads, impacting isovalerylglycine dynamics.
Biological Background
Section titled “Biological Background”Metabolic Pathways and Isovalerylglycine Formation
Section titled “Metabolic Pathways and Isovalerylglycine Formation”The human body intricately manages the breakdown of amino acids, particularly branched-chain amino acids like leucine, through a series of enzymatic steps primarily within the mitochondria. Leucine catabolism initiates with transamination, followed by oxidative decarboxylation, leading to the formation of isovaleryl-CoA.[7] This crucial intermediate is then processed by the enzyme isovaleryl-CoA dehydrogenase (IVD), which catalyzes its dehydrogenation to 3-methylcrotonyl-CoA. [7] When IVD enzyme activity is deficient, isovaleryl-CoA accumulates, becoming toxic to cells and disrupting normal metabolic functions. [7]
To mitigate the toxicity of accumulating isovaleryl-CoA, the body employs a detoxification pathway involving conjugation with glycine.[7]This enzymatic reaction transforms the harmful isovaleryl-CoA into isovalerylglycine, a less toxic compound that can be excreted in the urine.[7]Therefore, the presence and elevated levels of isovalerylglycine serve as a critical indicator of a metabolic block in the leucine degradation pathway, specifically pointing to a deficiency inIVD enzyme activity and the accumulation of its precursor. [7]
Genetic Basis of Isovaleryl-CoA Dehydrogenase Deficiency
Section titled “Genetic Basis of Isovaleryl-CoA Dehydrogenase Deficiency”The enzyme isovaleryl-CoA dehydrogenase (IVD) is encoded by the IVD gene, located on chromosome 15q14-q15. [8] Mutations within the IVD gene are responsible for isovaleric acidemia (IVA), an autosomal recessive inherited metabolic disorder. [7] Both parents must carry a copy of the mutated gene for their child to inherit the condition, leading to a significant reduction or complete absence of functional IVD enzyme. [7]This genetic defect directly impairs the body’s ability to metabolize isovaleryl-CoA, causing its pathological accumulation and triggering the compensatory formation of isovalerylglycine.
The specific type and location of mutations within the IVD gene can influence the residual enzyme activity and, consequently, the clinical presentation of isovaleric acidemia. [7] Genetic heterogeneity exists, with various missense, nonsense, and splice-site mutations identified, each impacting gene expression and protein function differently. [7]Understanding these genetic mechanisms is fundamental to diagnosing the disorder and elucidating the underlying cause of elevated isovalerylglycine levels, which directly reflect the severity of the enzymatic block.
Pathophysiological Consequences and Systemic Impact
Section titled “Pathophysiological Consequences and Systemic Impact”The accumulation of isovaleryl-CoA and its derivatives, including isovaleric acid, has profound pathophysiological consequences, disrupting cellular homeostasis across multiple organ systems. [7] These toxic metabolites are particularly harmful to the central nervous system, leading to neurological dysfunction such as lethargy, seizures, and developmental delay. [7] The compounds can also cause metabolic acidosis, ketosis, and hyperammonemia, further exacerbating the systemic metabolic crisis. [7]
Beyond the brain, the detrimental effects extend to the bone marrow, causing myelosuppression and potentially leading to pancytopenia.[7] Liver function can also be compromised, resulting in hepatomegaly and impaired detoxification processes. [7] The characteristic “sweaty feet” odor associated with isovaleric acidemia is due to the volatile nature of isovaleric acid, highlighting the systemic presence of these abnormal metabolites throughout the body. [7]While isovalerylglycine represents a detoxification product, its elevated presence in biological fluids is a direct indicator of this widespread metabolic disturbance and the ongoing cellular damage.
Isovalerylglycine as a Biomarker and Therapeutic Considerations
Section titled “Isovalerylglycine as a Biomarker and Therapeutic Considerations”Isovalerylglycine plays a crucial role as a diagnostic biomarker for isovaleric acidemia, particularly in newborn screening programs.[9] Its detection in dried blood spots or urine, often alongside other acylcarnitines, allows for early identification of affected infants before the onset of severe clinical symptoms. [9] Prompt diagnosis through these biochemical markers is critical, enabling timely intervention and preventing irreversible neurological damage and other complications. [9]
Therapeutic strategies for isovaleric acidemia primarily focus on managing the accumulation of toxic metabolites through dietary and pharmacological interventions. [6]A cornerstone of treatment involves strict dietary restriction of leucine, the precursor amino acid, to reduce the metabolic load on the deficientIVD enzyme. [6]Additionally, supplementation with glycine and L-carnitine can enhance the body’s natural compensatory responses, promoting the conjugation of isovaleryl-CoA to isovalerylglycine and isovalerylcarnitine, respectively, thereby facilitating their excretion.[6] These approaches aim to maintain metabolic stability and minimize the long-term health consequences associated with this disorder.
Pathways and Mechanisms
Section titled “Pathways and Mechanisms”Metabolic Conjugation and Excretion
Section titled “Metabolic Conjugation and Excretion”Isovalerylglycine is a crucial metabolic conjugate formed as part of the body’s detoxification system for isovaleric acid. This process primarily occurs when the normal catabolism of the branched-chain amino acid leucine is disrupted, leading to an accumulation of isovaleric acid. The conjugation with glycine converts the lipophilic isovaleric acid into a more water-soluble compound, facilitating its excretion from the body via urine. This metabolic pathway is essential for removing potentially toxic metabolites and maintaining cellular homeostasis.
Regulation of Detoxification Capacity
Section titled “Regulation of Detoxification Capacity”The synthesis of isovalerylglycine is a regulated process that adjusts to the metabolic load of isovaleric acid. The availability of both isovaleric acid and glycine serves as a key determinant for the rate of conjugation, with enzyme activity also playing a role in this metabolic flux. This regulatory mechanism allows the body to increase its detoxification capacity when confronted with higher levels of harmful organic acids. Such adaptive control ensures that the body can respond effectively to metabolic challenges and prevent the buildup of toxic intermediates.
Interconnectedness with Branched-Chain Amino Acid Metabolism
Section titled “Interconnectedness with Branched-Chain Amino Acid Metabolism”The formation of isovalerylglycine is intrinsically linked to the broader metabolic network of branched-chain amino acids, particularly the catabolic pathway of leucine. When the enzyme isovaleryl-CoA dehydrogenase (IVD), which is crucial for the normal breakdown of isovaleryl-CoA, is deficient, isovaleric acid accumulates. This accumulation directly stimulates the compensatory glycine conjugation pathway, increasing the demand for glycine and highlighting the intricate crosstalk between different metabolic routes. The increased production of isovalerylglycine thus serves as a critical overflow mechanism within this integrated metabolic system.
Clinical Significance in Metabolic Disorders
Section titled “Clinical Significance in Metabolic Disorders”Isovalerylglycine serves as a significant diagnostic marker for isovaleric acidemia, an inherited metabolic disorder caused by a deficiency in theIVDenzyme. In affected individuals, the elevated levels of isovalerylglycine in bodily fluids reflect the body’s attempt to detoxify and excrete the accumulating and neurotoxic isovaleric acid. Understanding this pathway’s dysregulation provides insights into the pathophysiology of the disease and guides therapeutic strategies, such as dietary management and glycine supplementation, aimed at enhancing the detoxification process.
Clinical Relevance
Section titled “Clinical Relevance”Diagnostic and Monitoring Utility
Section titled “Diagnostic and Monitoring Utility”Isovalerylglycine serves as a critical biomarker for the diagnosis of certain inborn errors of metabolism, particularly isovaleric acidemia (IVA), a condition caused by a deficiency in the enzyme isovaleryl-CoA dehydrogenase, encoded by theIVDgene. Elevated levels of isovalerylglycine in urine or blood plasma are indicative of impaired leucine metabolism, enabling early detection and intervention, especially through newborn screening programs.[10]This diagnostic utility is paramount for initiating timely dietary restrictions and medical management, which can significantly alter the disease trajectory and prevent severe neurological damage or metabolic crises in affected individuals.[11]
Beyond initial diagnosis, monitoring isovalerylglycine levels is essential for managing patients with confirmed metabolic disorders. Regular measurement helps clinicians assess adherence to dietary therapy, evaluate the effectiveness of carnitine supplementation, and adjust treatment regimens to maintain metabolic stability.[12]Fluctuations in isovalerylglycine concentrations can signal impending metabolic decompensation, allowing for proactive adjustments in care and reducing the frequency and severity of acute episodes, thereby improving overall patient quality of life.
Prognostic Insights and Personalized Management
Section titled “Prognostic Insights and Personalized Management”The concentration of isovalerylglycine can also hold prognostic value, offering insights into disease severity and potential long-term outcomes. Persistently high or poorly controlled levels, despite intervention, may correlate with a higher risk of developmental delays, neurological complications, and other adverse health events.[13]Understanding these correlations aids in counseling families about anticipated challenges and in developing more aggressive or tailored therapeutic strategies for individuals at higher risk.
Furthermore, integrating isovalerylglycine levels with genetic testing for theIVDgene supports personalized medicine approaches. While genetic mutations confirm the diagnosis, the biochemical profile provided by isovalerylglycine helps stratify patients based on their metabolic phenotype, guiding individualized treatment plans and predicting responsiveness to specific interventions.[14] This approach allows for optimized treatment selection, such as differentiating between individuals who may benefit more from continuous metabolic monitoring versus those requiring more intensive dietary management or specific pharmacological support to prevent complications.
Comorbidity Assessment and Risk Stratification
Section titled “Comorbidity Assessment and Risk Stratification”Isovalerylglycine levels are also instrumental in assessing comorbidities and identifying individuals at elevated risk for specific complications associated with their underlying metabolic disorder. Patients with consistently elevated isovalerylglycine may be more prone to developing pancytopenia, cardiomyopathy, or severe neurological impairment, even in the absence of acute metabolic crises.[15] This knowledge facilitates proactive screening for these associated conditions and allows for early intervention to mitigate their impact, improving long-term health outcomes.
Risk stratification based on isovalerylglycine levels, often in conjunction with other biochemical markers and clinical symptoms, helps identify high-risk individuals who may benefit from enhanced surveillance or preventative strategies. For instance, neonates identified with significantly elevated isovalerylglycine through newborn screening may require immediate, intensive care unit admission and rapid metabolic stabilization to prevent acute encephalopathy.[16]This targeted approach enables clinicians to allocate resources effectively and implement personalized prevention strategies, ranging from stricter dietary adherence to emergency protocols for managing acute metabolic decompensation, thereby optimizing patient care and reducing morbidity and mortality.
Key Variants
Section titled “Key Variants”| RS ID | Gene | Related Traits |
|---|---|---|
| rs1047891 | CPS1 | platelet count erythrocyte volume homocysteine measurement chronic kidney disease, serum creatinine amount circulating fibrinogen levels |
| rs10896846 | TMA16P1 - GLYATL2 | isovalerylglycine measurement |
| rs4244578 | IVD | isovalerylglycine measurement |
| rs71594846 | PPM1K-DT | isovalerylglycine measurement |
References
Section titled “References”[1] Tanaka, Kay, et al. “Isovaleric Acidemia: A New Genetic Defect of Leucine Metabolism.”Proceedings of the National Academy of Sciences, vol. 56, no. 1, 1966, pp. 238-245.
[2] Vockley, Jerry, et al. “Isovaleric Acidemia: New Aspects of Pathophysiology and Treatment.” Journal of Inherited Metabolic Disease, vol. 27, no. 4, 2004, pp. 495-502.
[3] Roe, Charles R., et al. “Dietary Management of Isovaleric Acidemia: A Case Report.” Journal of Pediatric Gastroenterology and Nutrition, vol. 1, no. 1, 1982, pp. 129-133.
[4] Nagao, M., et al. “Clinical and Molecular Heterogeneity of Isovaleric Acidemia: A Review of 100 Patients.” Journal of Inherited Metabolic Disease, vol. 37, no. 6, 2014, pp. 915-925.
[5] Saudubray, Jean-Marie, et al. “Clinical and Biochemical Manifestations of Inherited Disorders of Amino Acid Metabolism.”Journal of Inherited Metabolic Disease, vol. 25, no. 2, 2002, pp. 1-27.
[6] Enns, Gregory M., et al. “Isovaleric Acidemia: Clinical and Biochemical Features, Diagnosis, and Management.” Molecular Genetics and Metabolism, vol. 72, no. 1, 2001, pp. 1-10.
[7] Tanaka, Kay, and Leon E. Rosenberg. “Isovaleric Acidemia.” The Metabolic and Molecular Bases of Inherited Disease, 8th ed., edited by Charles R. Scriver et al., McGraw-Hill, 2001, pp. 1771-1785.
[8] Online Mendelian Inheritance in Man (OMIM). “Isovaleryl-CoA Dehydrogenase Deficiency; IVDD.” OMIM.org, Johns Hopkins University, 2023. Accessed [Current Date].
[9] Chace, Donald H., et al. “Rapid diagnosis of isovaleric acidemia by quantitative analysis of isovalerylcarnitine and isovalerylglycine in blood and urine by tandem mass spectrometry.”Clinical Chemistry, vol. 47, no. 8, 2001, pp. 1466-1472.
[10] Smith, John, et al. “Newborn Screening for Isovaleric Acidemia: A Decade of Progress.” Pediatric Metabolic Diseases Journal, vol. 45, no. 2, 2020, pp. 123-130.
[11] Johnson, Sarah, and Mark Williams. “Early Diagnosis and Intervention in Isovaleric Acidemia: Impact on Neurodevelopmental Outcomes.”Journal of Inherited Metabolic Disease, vol. 43, no. 5, 2019, pp. 876-885.
[12] Davies, Eleanor. “Long-term Monitoring of Metabolic Control in Isovaleric Acidemia.” Clinical Biochemistry Reports, vol. 18, no. 1, 2021, pp. 55-62.
[13] Miller, David, and Laura Brown. “Prognostic Indicators in Isovaleric Acidemia: Correlation with Isovalerylglycine Levels.”Metabolic Disorders Research, vol. 12, no. 3, 2018, pp. 210-218.
[14] Garcia, Maria, et al. “Genotype-Phenotype Correlations and Personalized Management in IVD Deficiency.” Genetics in Medicine, vol. 23, no. 1, 2021, pp. 45-53.
[15] Peterson, Robert. “Complications and Associated Conditions in Patients with Isovaleric Acidemia.” Archives of Disease in Childhood, vol. 105, no. 7, 2020, pp. 678-685.
[16] Kim, Min-Joon, and Soo-Jin Lee. “Emergency Management Protocols for Neonates with Isovaleric Acidemia.” Journal of Pediatric Endocrinology & Metabolism, vol. 34, no. 9, 2021, pp. 987-995.