on-line at: www.actabp.pl A focus on homocysteine in autism

Homocysteine is an amino acid, which plays several important roles in human physiology. A wide range of disorders, including neuropsychiatric disorders and autism, are associated with increased homocysteine levels in biological fluids. Various B vitamins: B6 (pyridoxine), B12 (cobalamin), and B9 (folic acid) are required as co-factors by the enzymes involved in homocysteine metabolism. Therefore, monitoring of homocysteine levels in body fluids of autistic children can provide information on genetic and physiological diseases, improper lifestyle (including dietary habits), as well as a variety of pathological conditions. This review presents information on homocysteine metabolism, determination of homocysteine in biological fluids, and shows abnormalities in the levels of homocysteine in the body fluids of autistic children.


INTRODUCTION
Homocysteine is a non-protein, naturally occurring amino acid of potential clinical significance.It is a homologue of the amino acid cysteine (Fig. 1, Table 1).
Homocysteine was obtained in 1935 by the reduction of homocysteine with metallic sodium in ammonia (Rigel B, duVigneaud V, JBC 1935).The relationship between different factors (genetic, physiological, lifestyle, as well as a variety of pathological conditions) and the level of homocysteine in plasma and urine was the subject of numerous studies.Homocystinuria, an inherited disease being the result of homozygous cystathionine-βsynthase (CBS) deficiency and characterized by episodes of thromboembolism, as well as mental retardation, hepatic steatosis, lens dislocation, and osteoporosis (Malinowska et al., 2012), was recognized as a risk factor for human diseases and described in 1960s by Carson and Niell, who found large quantities of homocysteine in the urine of siblings with mental retardation (Carson & Niell, 1962).At the same time, during biochemical investigations of children with coşanomalies, homocystinuria was described by Gerritsen et al. (Gerritsen et al., 1962;Gerritsen & Waisman, 1964).In 1964 Mudd et al. (1964) demonstrated that homocystinuria is caused by the deficiency or absence of cystathionine synthase.In 1969, McCully noted a link between the elevated levels of homocysteine in blood and atherosclerosis (McCully, 1969).Since the early 1990s homocysteine has become the subject of a growing number of worldwide studies and the leading theme of many international conferences.The mainstream of research examines the impact of elevated levels of homocysteine in cardiovascular (Jacobsen, 1998;Auer et al., 2001) and neural tube defects (Nelen, 2001;Hague, 2003), mental illness, and cognitive impairment (Seshadri et al., 2002;Nilsson et al., 2002).Homocysteine is a sensitive indicator of subclinical folate or vitamin B12 deficiencies, rather than a standard hematologic indicator (Duthie et al., 2002).For example, in uremic patients without other evidence of folate deficiency, folate supplementation reduced the degree of hyperhomocysteinemia, but did not consistently restore the level of homocysteine to its normal value (Thambyrajah et al., 2000;Perna et al., 2005).The connection between autism spectrum disorders (ASD) and hyperhomocysteinemia was presented for the first time by Paşca in 2006 (Paşca et al., 2006).
In the human body, homocysteine is metabolised through two B-vitamin-dependent pathways: transsulfuration to cysteine, or remethylation to methionine (Perla-Kajan et al., 2007).Homocysteine metabolism also leads to the formation of a highly reactive compound Hcy-thiolactone (HTL), the synthesis of which is connected with the misactivation of homocysteine by aminoacyl-tRNA synthetase (AARS) or erroneous activation (Jakubowski, 1990).

THE STATUS OF THE DIETARY AND LIFESTYLE FACTORS AND THE LEVEL OF HOMOCYSTEINE IN PLASMA
The concentration of homocysteine in body fluids depends on many factors: age, sex, diet, use of certain drugs, abuse of caffeine, alcohol, smoking, and in the course of certain diseases such as kidney failure and cancer (Table 2).
Because of the association of the levels of plasma homocysteine with B vitamins and folates intake, abnormal levels of plasma homocysteine are considered as a marker of the nutritional status.Consumption of fruit and vegetables rich in folates lowers the levels of homocysteine in plasma (Kawashima et al., 2007).Foods with naturally occurring folates are better at reducing the levels of homocysteine in blood than synthetic folic acid (Bogers et al., 2007).Synthetic folic acid has nearly double the bioavailability as folates from food.Folic acid, folinic acid (formyltetrahydrofolate), and 5-methyltetrahydrofolate constitute the major circulating forms of folates.Vitamins B12 and B6 are co-factors for enzymes involved in remethylation and transsulferation, respectively.The most preferred by clinicians form of vitamin B12 is methylcobalamin.In literature, cyanocobalamin and pyridoxine hydrochloride are the most often mentioned forms of vitamins B12 and B6 (Guilliams, 2004).One of the lifestyle factors which increases the level of homocysteine in plasma is coffee.There is no relationship between the consumption of decaffeinated coffee and plasma homocysteine levels (Grubben et al., 2000).Coffee affects the metabolism of homocysteine in just a few hours after intake, and its effect is significant even after an overnight fast (Verhoef et al., 2002).Another lifestyle factor that influences the level of homocysteine in blood is alcohol (excluding beer) (van der Gaag et al., 2000).In case of a single instance of alcohol consumption among healthy individuals resulting in intoxication, plasma homocysteine is not increased significantly.Moderate beer consumption does not contribute to the changes in the levels of plasma homocysteine, however, there is a positive association between drinking spirits and wine and the level of homocysteine (Lutz, 2008).Another factor which affects the level of homocysteine in plasma is smoking.Among smokers, low levels of vitamins B6, B12, and folate are observed, as well as elevated levels of homocysteine in plasma (O'Callaghan et al., 2002).

GENETIC ASPECT OF HOMOCYSTEINE
The study of genetic defects associated with Hcy metabolic pathways is an important aspect of hyperhomocysteinemia.In this regard, the status of the C677T variant of the methylenetetrahydrofolate reductase (MTHFR) gene is very important.The MTHFR enzyme regulates folate availability and is responsible for the conversion of 5,10-methylenetetrahydrofolate to the 5-methyltetrahydrofolate (MTHF or 5MTHF).Codon 677 of the MTHFR gene (677C→T) causes a decrease in the activity of the MTHFR enzyme by 60%, and an increase in the levels of plasma homocysteine levels, especially in people with low levels of B vitamins (Weisberg et al., 1998;Hustad et al., 2007).The 677C→T is associated with neural tube defects, cerebrovascular and cardiovascular diseases (Nakata et al., 1998;Reinhardt et al., 1998), as well as with psychiatric disorders (Bonig et al., 2003;Bjelland et al., 2003).Another important genetic aspects of hyperhomocysteinemia are cystathionine β-synthase (CBS) gene mutations.The CBS enzyme catalyzes a re- action that permanently removes homocysteine from the methionine pathway by diverting it to the transsulfuration pathway, where vitamin B6 is needed as a co-factor.It was reported that there are associations between autism and functional gene variants within the B vitamindependent folate, methionine, and transmethylation pathways (Adams et al., 2007;Boris et al., 2007;James et al., 2006;James et al., 2010).Moreover, there are data that use of prenatal vitamins may reduce the risk of having children with autism (Schmidt et al., 2011).Genetic alterations of enzymes such as the CBS or MTHFR have an effect on the increases of Hcy-thiolactone.Hcy-thiolactone damages proteins which may underlie the Hcy-associated cardiovascular disease in humans (Jakubowski et al., 2001;Jakubowski et al., 2009).The studies of genetic hyperhomocysteinemia in humans in animal models show that homocysteine has an important impact on atherothrombosis.Chwatko et al. (2007) presented that Hcy-thiolactone levels are elevated in hyperhomocysteinemic mice.They discovered that plasma Hcy-thiolactone is elevated 59-fold in human patients with hyperhomocysteinemia secondary to mutations in methylenetetrahydrofolate reductase and 72-fold cystathionine-synthase genes, respectively.Ramaekers et al. (2002) presented research on children with low levels of 5-methyltetrahydrofolate in the cerebrospinal fluid.These children above the age of 4 to 6 months had neurological regression.Supplementation with folic acid eliminated the symptoms.Schmidt et al. (2011) shows a theory that children whose mothers have MTHFR 677 TT, CBS genotypes and do not report consuming prenatal vitamins have an increased risk of autism disorder.

DETERMINATION OF HOMOCYSTEINE IN BIOLOGICAL FLUIDS
Several methods are used to measure the level of homocysteine.As corrently pointed out in the work by Sawuła et al. (2008), due to the fact that a small increase in Hcy levels can constitute a risk factor for the cardiovascular disease, it is extremely important in the studies of Hcy levels to select a highly precise and accurate method.Such values are characterized by methods based on chromatography.
Among the remaining separation methods, chromatographic techniques are the most useful and often applied: ion-exchange chromatography equipped with an amino acid analyzer, high-performance liquid chromatography (HPLC) with electrochemical, fluorescent, or UV detection (House et al., 2000;Shoveller et al., 2004;Sawuła et al., 2008;Sawuła et al., 2009), liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Rafii et al., 2007), capillary electrophoresis (Caussé et al., 1999) and gas chromatography-mass spectrometry (GC-MS) (Kałużna-Czaplińska et al., 2011a).All these methods have their advantages and limitations related to sensitivity, selectivity, samples derivatization, analysis reproducibility, measurement and equipment costs etc.The choice of the method depends mainly on its availability and the particular need in analysis.

VITAMIN DEFICIENCY, SUPPLEMENTATION, AND AUTISM
In recent years, abnormalities in the levels of amino acids in the human body have been identified in children with different disorders and autism (Bongiovanni & Feinerman, 2003;James et al., 2004).Amino acids, including homocysteine, are expelled in urine and plasma, and provide essential information on the diet and functioning of the alimentary system.Several studies on the neurotoxicity of homocysteine indicate that Hcy can induce neuronal damage and cell loss through excitotoxicity as well as apoptosis (Sawada et al., 1982;Lee et al., 1988;Lipton et al., 1997;Kruman et al., 2000).One of the reasons for this state may be the failure of cerebral tissue to metabolize Hcy by the betaine and transsulfuration pathways, resulting in the accumulation of Hcy in the nervous system (Finkelstein, 1998).
Many studies have reported the beneficial effects of vitamin and nutritional supplements in the treatment of autism spectrum and other neurological disorders.Monitoring of homocysteine levels in various body fluids gives information about possible deficiencies of various B vitamins: B6, B12, and folic acid (Nekrassova et al., 2003).Refsum et al. (2004) discussed the application of total homocysteine (tHcy) measurements in the diagnosis of folate and vitamin B12 deficiencies in various psychiatric and neurologic disorders.High levels of homocysteine and oxidative stress are generally associated with neuropsychiatric disorders such as autism (Chauhan & Chauhan, 2006;Suh et al., 2008).Homocysteine is a powerful excitotoxin, and its metabolism products cause protein damage and structural changes which generate toxic proteins and induce an autoimmune response (Jakubowski & Głowacki, 2011).
Table 3 shows the comparison of the level of homocysteine, folate and vitamin B12 in biological fluids between autistic children and control children.Paşca et al. (2006) were the first who discovered the relationship between the levels of homocysteine in serum of autistic children and vitamin B12 deficiency.They found out that high levels of homocysteine and oxidative stress markers are associated with autism.Their studies showed statistically significant differences in tHcy levels and arylesterase activities of human paraoxonase 1 (PON1) in children with autism compared to the control group.Kałużna-Czaplińska et al., (2009) identified potential nutritional deficits in children with autism, and the relationship between the diet and functioning of an autistic child.The analysis of daily intake of nutrients by autistic children based on a 7-day diet and their parents' reports shows that autistic children have deficiencies of vitamins B6, B9, B12, and C caused by improper diets.The characteristics of the functioning of autistic children based on psychological tests and speech therapy were also analyzed.The studies showed that the diet of autistic children should be enriched in vitamins of group B and vitamin C. Such an approach was rationalized by systematic studies based on the analysis of homocysteine in the urine of autistic children performed by GC-MS method (Kałużna-Czaplińska et al., 2011a).The levels of homocysteine in urine samples of autistic children were significantly higher than those of healthy children.The results were very helpful in preparing and determining individual diets for autistic children.It appeared that vitamin supplementation reduces the levels of homocysteine in the urine of autistic children (Kałużna-Czaplińska et al., 2011b).Moreover, the results showed that a dietary intake of vitamins B6, B12, and folic acid is more effective than the intake of vitamin B6 and B12 alone.The results lead to the recommendation of supplementing the diets of autistic children with vitamins B6, B12, and folic acid.Similar conclusions come from the paper of Ali et al. (2011).Supplementary intake of vitamins B6, B12, and folate was effective in lowering the levels of blood homocysetine.Previous studies from 1981 (Lowe et al. 1981) showed that the level of B vitamins in biological fluids of autistic children and the levels of serum folate and vitamin B12 are congruous in autistic children and the control group.These findings may result from the fact that more than half of the autistic children were treated with daily vitamin supplements containing B vitamins.James et al. (2004) investigated the abnormal metabolism of methionine and homocysteine and also presented the congruous level of Hcy in the plasma of autistic children in comparison with the control group.The decreased capacity for methylation associated with low levels of plasma homocysteine in children with autism was related to the fact that 80% of autistic children were taking folic acid and vitamin B12 supplements prior to the beginning of the study.Such a treatment decreased the level of homocysteine and resulted in almost the same level of homocysteine in the plasma of autistic children and the control group.
The fact is that nutritional supplements like B vitamins should not be the primary source of nutrition but sometimes it is difficult to get enough B vitamins from food.Waśkiewicz et al. (2007) presented the results of an inadequate intake of vitamins B6, B12, and folate in Polish population.49 to 84% of the population includes the recommended amounts of vitamins B6, B12, and folic acid in their daily diet, while the recommended daily quantity of folic acid is taken only by 10% of women and 22% of men.Also, it is very difficult to maintain an adequate intake of B vitamins with a balanced diet for some groups like vegetarians, and it is a big problem in countries with no fortification of foods with B vitamins.It is especially difficult to get all the necessary B vitamins from food; therefore supplements are essential (Finglas et al., 2006).
According to the studies conducted by Xia et al. (2010), dietary habits of autistic children shows that most of them had inadequate intake of folic acid, vitamins B6, A, C, and zinc.Dietary deficiency of vitamin B12 was identified as the cause of partially reversible optic neuropathy in 3 autistic children (Pineles et al., 2010).Treatment with vitamin B12 and normalization of vita-min B12 levels resulted in the improvement of visual functioning of children.These investigations show that food selectivity and vitamin deficiency cause visual loss and optic atrophy.According to another report (Paul et al., 2007), in the case of some autistic children vitamin deficiencies are caused by improper dietary habits and increased food selectivity.Deficiencies in folic acid as well as vitamins B6 and B12 in the nutrition result in higher levels of homocysteine and an increase in some autistic symptoms (Williams et al., 2005).The theory of efficacies of nutritional supplements of vitamin B6 in autism-spectrum disorders was presented by Adams et al. (2004).His paper shows statistically significant improvements in sleep and gastrointestinal problems in autistic children compared to the placebo group.Also, Xia (2011) described a case of a 9-year-old boy with autism who responded positively to vitamin B6 nutritional supplements.This diet resulted in the improvement of communication, sociability, cognitive awareness, and behavior.
The studies suggest a very important relationship between high levels of homocysteine and deficiencies of vitamins B: B6, B12, and folic acid for the proper functioning of autistic children.The higher level of homocysteine in the body fluids can be used as a marker of possible nutritional deficits of B vitamins.

CONCLUSIONS
The high level of serum and urinary homocysteine is associated with pathophysiology of autism spectrum disorders and may serve as a diagnostic tool for the detection of nutrient deficiencies in the case of autistic children.However, it should be very carefully considered whether abnormal levels of homocysteine are the result of nutritional deficiencies in children or they result from genetic and physiological diseases or other pathological conditions.
A recent report has revealed that folate or methionine metabolism is altered in autistic children.The cycle of folate-methionine can play a key role in the etiology of autism.There is a need to confirm whether the supplementation of the deficits in folate and vitamins B6 and B12 in the metabolism of autistic children leads to behavioral and functional benefits.

•
It is a non-protein amino acid.• It is a homologue of the amino acid cysteine, differing by an additional methylene (-CH 2 -) group.• Homocysteine is not obtained from the diet.• Homocysteine can be recycled into methionine or converted into cysteine with the aid of B-vitamins.• Deficiencies of vitamins: folic acid (B9), pyridoxine (B6), or B12 (cobalamin) lead to high levels of homocysteine.