Breifly describe the methods ( design, participants, materials, procedure, what was manipulated, what was measures, how data were analyzed.

© 2010 El-Ansary et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

Open Access Journal of Clinical Trials 2010:2 49–57

Open Access Journal of Clinical Trials

49

O R I G I N A L R E S E A R C H

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Activities of key glycolytic enzymes in the plasma of Saudi autistic patients

A El-Ansary1

S Al-Daihan1

A Al-Dabas1

L Al-Ayadhi2

1Biochemistry Department, Science College, 2Autism Research and Treatment Unit, Department of Physiology, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia

Correspondence: Afaf El-Ansary Biochemistry Department, Science College, King Saud University, P.O Box 22452, Riyadh, 11495, Saudi Arabia Tel +9614682184 Fax +9614769137 Email afafelansary@yahoo.com; elansary@ksu.edu.sa

Objective: Measurement of plasma levels of lactate, lactate oxidase (LOX), pyruvate kinase (PK), and hexokinase (HK) as possible glycolytic parameters to assess brain damage in autistic patients.

Design and methods: Plasmatic levels of lactate, LOX, PK, and HK were determined in 20 autistic children aged 3–15 years and 20 age-matching healthy control subjects.

Results: Plasmatic levels of lactate and LOX were significantly higher in autistic patients compared to healthy subjects and that of PK and HK were significantly lower in these patients

as compared to controls. This could reflect the impaired metabolism of astrocytes, the brain

cells responsible for the production and provision of lactate, as the primary metabolic fuel for

neurons.

Conclusion: Remarkably different levels of plasma glycolytic parameters were recorded in Saudi autistic patients. This could be correlated to the impairment of energy metabolism,

glutathione depletion, and lead intoxication previously detected in the same investigated samples.

The identification of biochemical markers related to autism would be advantageous for earlier

clinical diagnosis and intervention.

Keywords: autism, glycolysis, lactate, lactate oxidase, pyruvate kinase, hexokinase

Introduction Autism is a disorder of reciprocal social interaction, behavioral repertoire, and language

and communication disabilities.1 Because the phenotype ranges from manifest disability

to specific performance elevation, the term autistic spectrum disorder (ASD) is now

commonly used to denote the Diagnostic and Statistical Manual of Mental Disorder,

4th Edition (DSM-IV)-defined group of pervasive neurodevelopmental disorders

encompassing autistic disorder including Asperger’s disorder, Rett’s disorder, and

pervasive developmental disorder not otherwise specified (PDDNOS).1,2 A fraction

of cases have a defined genetic cause, but the apparent increase in prevalence of ASD

as reviewed is suggestive of an environmental contribution.3–5 Changes in awareness

and diagnostic criteria may explain some of the rise but a true increase in prevalence

has not been excluded.6,7 Elevated ASD rates in urban versus rural areas are consistent

with an environmental contribution.8,9 Recently, Weissman and colleagues pointed

to several underlying pathophysiological mechanisms in autism, including altered

neurite morphology, synaptogenesis and cell migration due to abnormalities in distinct

ensembles of proteins and pathways. In a cohort analysis, they reported that defective

mitochondrial oxidative phosphorylation is an additional pathogenetic basis for a subset

of individuals with autism.10 Impairment of energy metabolism due to mitochondrial

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dysfunction was confirmed by Al-Mosalem and colleagues

in a study of 30 Saudi autistic children.11

Glucose had long been thought to fuel oxidative metabo-

lism in active neurons until the recently proposed astrocyte-

neuron lactate shuttle hypothesis (ANLSH) challenged this

view. According to the ANLSH, activity-induced uptake of

glucose takes place predominantly in astrocytes, which metab-

olize glucose anaerobically. Lactate produced from anaerobic

glycolysis in astrocytes is then released from astrocytes and

provides the primary metabolic fuel for neurons. The conven-

tional hypothesis asserts that glucose is the primary substrate

for both neurons and astrocytes during neural activity and

that lactate produced during activity is removed mainly after

neural activity.12 The dependence of brain function on blood

glucose as a fuel does not exclude the possibility that lactate

within the brain might be transferred between different cell

types and serve as an energy source. It has been suggested

recently that 1) about 85% of glucose consumption during

brain activation is initiated by aerobic glycolysis in astrocytes,

triggered by demand for glycolytically derived energy for

Na+ -dependent accumulation of transmitter glutamate and

its amidation to glutamine, and 2) the generated lactate is

quantitatively transferred to neurons for oxidative degrada-

tion. However, astrocytic glutamate uptake can be fueled by

either glycolytically or oxidatively-derived energy and the

extent to which “metabolic trafficking” of lactate might occur

during brain function is unknown.13 The subcellular compart-

mentalization of pyruvate allows neurons and astrocytes to

select between glucose and lactate as alternative substrates,

depending on their relative extracellular concentration and

the operation of a redox switch. This mechanism is based

on the inhibition of glycolysis at the level of glyceraldehyde

3-phosphate dehydrogenase by NAD (+) limitation. Follow- ing glutamatergic neurotransmission, increased glutamate

uptake by the astrocytes is proposed to augment glycolysis

and tricarboxylic acid cycle activity, balancing to a reduced

cytosolic NAD+/NADH in the glia. Reducing equivalents are

then transferred to the neuron resulting in a reduced neuronal

NAD+/NADH redox state. This may eventually switch off

neuronal glycolysis, favoring the oxidation of extracellular

lactate in the lactate dehydrogenase (LDH) equilibrium and

in the neuronal tricarboxylic acid cycles. Finally, pyruvate

derived from neuronal lactate oxidation, may return to the

extracellular space and to the astrocyte, restoring the basal

redox state and beginning a new loop of the lactate/pyruvate

transcellular coupling cycle.14

For some time, it has been known that in cultured astro-

cytes, nitric oxide (NO) can upregulate the rate of glucose

consumption and lactate production, suggesting glycolysis

activation, a phenomenon that is possibly a consequence of

the NO-mediated mitochondrial inhibition.15 However, it is

surprising that, in contrast to astrocytes, neurons do not dis-

play increased glycolytic rate upon mitochondrial inhibition

and this leads to neuronal cell death. Only astrocytes respond

by activating, very rapidly (ie, within a few minutes), the gly-

colytic pathway.16. Interestingly, the increased glycolytic rate

in astrocytes served to preserve cells from ATP depletion and

cell death, possibly because glycolytic ATP served to drive

the reverse activity of ATP synthase in order to maintain the

mitochondrial membrane potential.16. It is noteworthy that

in several neurodegenerative diseases, such as Alzheimers

or Huntingtons, decreased neuronal glycolytic activity has

been observed in neurons of the degenerating area.17,18 It is

therefore of interest to understand the mechanism(s) respon-

sible for the differential glycolytic response of astrocytes and

neurons upon mitochondrial inhibition.

ATP is considered to be a feed-back allosteric inhibitor of

6-phosphofructokinase 1 (Pkf1), key rate-limiting step in the

Fr eq

ue nc

y

5

4

3

2

1

0

Data

A B

1.631.501.381.251.131.000.880.750.630.50

Fr eq

ue nc

y

8

6

4

2

0

Data 3.503.002.502.001.501.000.50

Std. Dev = 0.33 Mean = 0.87 N = 15.00

Std. Dev = 0.82 Mean = 1.40 N = 15.00

Figure 1 A) Normal distribution of lactate in the control group. B) Normal distribution of lactate in the autistic group.

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glycolytic pathway.19 Accordingly, for many years it has been

considered that the decrease in cytosolic ATP concentrations

that follows mitochondrial dysfunction would stop Pfk1

inhibition, thus causing a rapid activation of glycolysis.

Indeed, inhibition of mitochondrial ATP synthesis which

triggers a rapid Pfk1 activation is known to take place in the

intact, but not disrupted, astrocytes.16 The involvement of

other glycolytic enzymes, such as hexokinase(s), pyruvate

cannot be disregarded as potential targets of NO-mediated

glycolysis activation.

Regarding the possibility of using plasma glycolytic

enzymes as biomarkers for brain damage, Tadeusz measured

the activities of several glycolytic enzymes such as hexokinase,

phosphofructokinase, pyruvate kinase, lactate dehydroge-

nase, as well as glycerol-1-phosphate dehydrogenase, and

(Mg2+)ATPase in normal cerebrospinal fluid (CSF) and blood

plasma. Samples were drawn from 12 healthy infants and in

supernatants from brain tissue slices taken during neurosurgi-

cal operations from infants of the same range of age.20 The val-

ues obtained confirm the high activity of the above-mentioned

enzymes in human brains and indicate an independence of this

activity in blood plasma and CSF. The origin of the activities

of the investigated enzymes in CSF seems to be mainly, if not

exclusively, from brain tissue. This finding might prove useful

for detection of brain tissue damage, as was earlier shown with

LDH activity in CSF and plasma.

Selakovi et al reported that many substances are released

into the CSF and blood during brain damage but the ideal

damage marker would have to satisfy certain requirements: to

be localized intracellularly, present in high concentration in

brain tissue, and to be relatively easy to detect. They reported

that neuron-specific enolase (NSE) as a glycolytic pathway

isoenzyme, specific for phosphoglycerate and phosphoenol-

pyruvate (2-phospho-D-glycerate- hydrolase, EC. 4.1.11)

has recently been recorded as brain damage marker.21 The

increase in concentration of NSE in CSF and plasma has

been detected in patients with brain ischemia and can be

significant in the early diagnosis of BI.22

This information initiated our interest in measuring

three glycolytic enzymes (hexokinase, pyruvate kinase and

lactate dehydrogenase) in Saudi autistic children compared

to healthy age-matching control subjects in a trial. Our aim

was to investigate a potential correlation between the activity

of these enzymes with the previously measured parameters

related to energy metabolism and oxidative stress in the same

investigated samples.11

Material and methods Patients and subjects The subjects enrolled in this study were 20 children with autism

(16 males and 4 females) ranging in age from 3–15 years, and

another 20 age-matching children (15 males and 5 females) as

a control group. The diagnosis of autism was made by child

neuropsychiatrists based on the criteria of autistic disorder

as defined in the DSM-IV.2 Complete diagnostic work-ups

including medical, neurological, psychiatric, and psychologi-

cal evaluations were done for all of the studied children with

autism. All were of good physical health and were not taking

any medications or nutrient supplements. Written consent was

obtained from the parents of each subject, according to the

guidelines of the ethical committee of King Khalid Hospital,

King Saud University, Riyadh, Saudi Arabia.

Blood samples After an overnight fast, 10 mL blood samples were collected

from both groups in test tubes containing heparin as an anti-

coagulant. Centrifugation was done at 3000 rpm. Plasma was

obtained and deep frozen (–80°C) until analysis time.

100.00

160.27

0.00 20.00 40.00 60.00 80.00

100.00 120.00 140.00 160.00 180.00

AutisticControl

Lactate determination

Figure 2 Percentage change of lactate determination in autistic group compared to control.

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Chemicals All chemicals used in this study were of analytical grade and

were products of Sigma (St. Louis, MO, USA), or Merck

(Darmstadt, Germany). Lactate oxidase and lactate kits were

products of the United Diagnostics Industry (UDI), Kingdom

of Saudi Arabia.

Biochemical analyses Measurement of lactate Lactate present in the samples was determined according to the

method of Brandt and colleagues using a diagnostic kit.23

Measurement of LOX Quantitative determination of LOX in plasma was performed

according to the method of Henry using a lactate to pyruvate

kinetic method.24

Measurement of HK Hexokinase was assayed in plasma according to the method

of Abraham-Neto and colleagues in which a reduction of

NADP+, through a coupled reaction with glucose-6-phosphate

dehydrogenase (G-6-PDH), is determined spectrophotometri-

cally by measuring the increase in absorbance at 340 nm.25

Measurement of PK Pyruvate kinase was determined in plasma according to

the method of Malcovati and Valentini by which the rate

of decrease in absorbption at 340 nm, due to oxidation of

NADH by coupling the system with an excess of LDH, was

followed.26

Statistical analysis SPSS software (SPSS Inc., Chicago, IL) was used to analyze

the data. Results were expressed as mean ± standard devia- tion (SD). The data from the patient group was compared

with data from the control group using Student’s t-test.

A P value of 0.05 was considered statistically significant.

Pearson correlations between the measured parameters are

presented.

Results Levels of lactate, LOX, PK and HK for both the control and

autistic groups are presented in Tables 1 and 2. Results are

given as Mean ±S.D. Normal distribution of the measured parameters in control and autistic Saudi patients together with

the percentage change of the measured parameters in autistic

compared to control subjects are presented in Figures 1–7.

Discussion The concept of brain injury is heterogeneous in terms of

etiology as well as type and severity of motor and associated

disabilities. At this point, because of the survival of extremely

premature infants and severely hypoxic neonates, the risk of

brain damage has not been eliminated. Lifelong disabilities

such as autism, cerebral palsy, epilepsy, behavioral and

learning disorders are still some of the consequences of brain

injury acquired in fetal life or the perinatal and neonatal

periods.27 Efforts to understand and prevent neonatal cerebral

injury are therefore worthwhile.

Finding a single biochemical marker which is both sensi-

tive and specific for brain injury is unlikely because the brain

contains many different types of cells, each with a different

threshold for injury and different sensitivities to various types

of injury. Because of the complexity of the brain, it may be

important to develop a panel of markers rather than a single

marker to be used as a screening tool. This panel would need

to include indicators of neuronal and glial cell injury, as well

as markers that are sensitive to direct trauma, hypoxia, and

oxidative stress.28

Evidence has accumulated over the last two decades

indicating that l-lactate (l-LAC) is an important cerebral

oxidative-energy substrate.29 The brain can take up l-LAC

from blood, particularly during intense exercise, as well as

in the initial minutes of recovery.30 Moreover, an “astro-

cyte-neuron l-LAC shuttle” has been proposed, in which

astrocytes take up glucose from blood, convert it into l-LAC

via glycolysis and then export l-LAC into the extracellular

phase through the isoform 1 of monocarboxylate transporter

(MCT1). In turn, neurons take up extracellular l-LAC via the

isoform 2 of monocarboxylate transporter (MCT2) and use

it as a fuel for mitochondrial respiration.31 Recently, it was

hypothesized that, in the brain, l-LAC is the principal product

of glycolysis, whether or not oxygen is present.32 The signifi-

cant increase of plasma lactate found in Saudi autistic children

involved in the present study compared to control subjects

could possibly reflect the impairment of neuron cell integrity

in these patients. This theory is supported by considering the

work of Al-Mosalim and colleagues which proposes there

is energy metabolism impairment in Saudi autistic children.

Table 1 Lactate levels in serum of control and autistic children Group N Mean ± SD Minimum Maximum P value

Control 15 0.872 ± 0.335 0.495 1.626  0.05

Autistic 15 1.398 ± 0.819 0.424 3.312

Note: This table describes the independent t-Test between the control and autistic groups in lactate determination levels.

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These authors encountered significantly higher activity levels

of Na+/K+ ATPase, creatine kinase, and NTPdase, together

with lactate in the plasma of autistic patients.11

Increased glycolysis confers adaptive advantages if it

allows the availability of excess pyruvate for lipid synthesis

or by providing essential anabolic substrates.33 Glucose

consumption through the pentose pathway may also provide

essential reduction equivalents (ie, NADPH) to decrease the

toxicity of reactive oxygen species conferring resistance to

senescence.34,35

Table 2 demonstrates the activity levels of lactate oxidase,

hexokinase, and pyruvate kinase are three critically important

glycolytic enzymes to monitor in Saudi autistic children as

compared to the control group. While lactate oxidase was

significantly elevated, both hexokinase and pyruvate kinase was

unexpectedly lower in the plasma of the autistic children. Fig-

ure 3b shows that 15 autistic patients had LOX activity higher

than 200 µmoles/minute/L compared to the control subjects represented in Figure 3a. This figure indicates that 11 out of the

15 control subjects had LOX activities less than 175 µmoles/ minute/L. Figure 4b illustrates that all the investigated sam-

ples from autistic children had PK activity levels less than

40 µmoles/minute/L while all the control subjects (Figure 4a) demonstrated significantly higher activities (ie, more than

65 µmoles/minute/L). A significantly lower activity of HK in autistic patients can be seen in Figure 5b in which 17

autistic exhibited HK activity levels less than 34 µmoles/ minute/L compared to significantly higher HK levels in the

control subjects (ie, more than 90 µmoles/minute/L). Lactate oxidase activation can be explained on the basis

of substrate availability, since lactate, as a substrate, was

found to be significantly higher in the plasma of the Saudi

autistic children compared to the healthy age-matched

control group. It is known that brain energy supply requires

the oxidative metabolism of glucose in mitochondria, and

when neural energy demands transiently exceed the rate

of oxidative metabolism, l-Lac is produced to supply

energy as a result of glycolytic processes.36,37 Increased

lactate level is related to the reduced use of pyruvate in

the citric acid cycle and the increase of anaerobic gly-

colysis. It is well known that oxidative stress increases

the concentrations of lactate dehydrogenase and thus

induces the increment of the lactate level.38 Therefore,

significantly increased lactate and LOX observed in the

present investigated samples (Figures 1 and 3) might

indicate the deficiency of mitochondria function or over-

expression of lactate oxidase in autistic children. This

explanation is supported by considering the work of

Table 2 Lactate oxidase (LOX), pyruvate kinase (PK), and hexokinase (HK) levels in plasma of control and autistic children Treatment Group N Mean ± SD Min Max P value

Lactate oxidase (LOX) (µ moles NAD reduced/min/L) Control 15 133.82 ± 60.56 82.40 266.00 0.05Autistic 24 237.71 ± 89.11 112.06 448.20

Pyruvate kinase (µ moles NADH oxidized/min/L) Control 11 73.84 ± 6.80 64.84 88.56 0.05Autistic 17 28.17 ± 5.55 20.50 38.54

Hexokinase (µ moles NAD reduced/min/L) Control 9 106.60 ± 16.28 88.56 138.30 0.05Autistic 17 25.22 ± 5.07 18.04 34.60

Note: This table describes the independent t-Test between the control and autistic groups in LOX, PK, and HK activity levels.

Data

275.0 250.0

225.0 200.0

175.0 150.0

125.0 100.0

75.0

Fr eq

ue nc

y

Data

Fr eq

ue nc

y

6

5

4

3

2

1

0

Std. Dev = 60.56 Mean = 133.8 N = 15.00

450.0400.0350.0300.0250.0200.0150.0100.0

7

6

5

4

3

2

1

0

Std. Dev = 89.11 Mean = 237.7 N = 24.00

A) B)

Figure 3 A) Normal distribution for control group in lactate oxidase (LOX) levels. B) Normal distribution for autistic group in lactate oxidase (LOX) levels.

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Al-Gadani and colleagues,39 and Al-Mosalim and

colleagues11 in which they recorded oxidative stress and the

disturbance of energy metabolism in Saudi autistic children

compared to age-matching control subjects.

Lower activities of HK and PK observed in this study

could reflect the less-adaptive capacity of autistic children to

cope with energy metabolism impairment, as was previously

documented by Al-Mosalim and colleagues.11. In addition,

glycolysis enhancement has recently been reported to

cooperate with autophagic mechanisms in preventing cas-

pase-independent cell death, further supporting the notion

that glycolysis activation is an important neuronal survival

pathway.40 The relationship between lower plasma HK and

PK reported in the present study and the lipoxidative stress

Data

Fr eq

ue nc

y

Data

Fr eq

ue nc

y

90.085.080.075.070.065.0

5

4

3

2

1

0

Std. Dev = 6.80 Mean = 73.8 N = 11.00

37.535.032.530.027.525.022.520.0

5

4

3

2

1

0

Std. Dev = 5.55 Mean = 28.2 N = 17.00

A) B)

Figure 4 A) Normal distribution for control group in pyruvate kinase levels. B) Normal distribution for autistic group in pyruvate kinase levels.

Data

Fr eq

ue nc

y

Data

A) B)

Fr eq

ue nc

y

140.0130.0120.0110.0100.090.0

3.5

3.0

2.5

2.0

1.5

1.0

.5

0.0

Std. Dev = 16.28 Mean = 106.6 N = 9.00

34.032.030.028.026.024.022.020.018.0

3.5

3.0

2.5

2.0

1.5

1.0

.5

0.0

Std. Dev = 5.07 Mean = 25.2 N = 17.00

Figure 5 A) Normal distribution for control group in hexokinase levels. B) Normal distribution for autistic group in hexokinase levels.

38.15 23.66

177.63

100.00

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

Pyruvate kinaseControl Lactate oxidase Hexokinase

Figure 6 Percentage change of lactate oxidase, pyruvate kinase, and hexokinase in autistic group compared to control.

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L ac

ta te

o xi

d as

e

y = 2.6766 × +144.22 R2 = 0.1061 P = 0.024

0

50

100

150

200

250

300

350

400

450

500

0 5 10 15 20 25 30 35 40 45

Lactate

A)

0 10 20 30 40

Pyruvate kinase

160

140

120

100

80

60

40

20

0

H ex

o ki

n as

e

50 60 70 80 90 100

y = 1.6516 × −19.728 R2 = 0.8935 P = 0.000

B)

0 50 100 150 200 Lactate oxidase

C) 100 90 80 70 60 50 40 30 20 10

0

P yr

u va

te k

in as

e

250 300 350 400 450 500

y = −0.1153 × +69.15 R2 = 0.2588 P = 0.006

160

140

120

100

80

60

40

20

0 0 50 100 150 200

D)

Lactate oxidase

H ex

o ki

n as

e

250 300 350 400 450 500

y = −0.1951 × +93.228 R2 = 0.2597 P = 0.008

Figure 7 A) Correlation between lactate and lactate oxidase (LOX) with best fit line curve (positive correlation). B) Correlation between pyruvate kinase and hexokinase with best fit line curve (positive correlation). C) Correlation between lactate oxidase and pyruvate kinase with best fit line curve (negative correlation). D) Correlation between lactate oxidase and hexokinase with best fit line curve (negative correlation).

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children who contributed their time and support to this study.

Thanks are extended to Dr Saba Abidi, assistant professor,

Biochemistry Department, King Saud University for her

effort in revising the English language of the manuscript.

Disclosures The authors report no conflicts of interest in this work.

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6. Croen LA, Grether JK, Hoogstrate J, Selvin S. The changing prevalence of autism in California. J Autism Dev Disord. 2002;32: 207–215.

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8. Palmer RF, Blanchard S, Stein Z, Mandell D, Miller C. Environmental mercury release, special education rates, and autism disorder: an eco- logical study of Texas. Health Place. 2006;12:203–209.

9. Williams JG, Higgins JP, Brayne CE. Systematic review of preva- lence studies of autism spectrum disorders. Arch Dis Child. 2006; 91:8–15.

10. Weissman JR, Kelley RI, Bauman ML, et al. Mitochondrial disease in autism spectrum disorder patients: a cohort analysis. PLoS One. 2008;3(11):3815.

11. Al-Mosalim O, El-Ansary A, Attas O, Al-Ayadhi L. Selected enzymes related to energy metabolism in Saudi autistic children. Clin Biochem. 2009;42:949–957.

12. Chih CP, Roberts Jr EL. Energy substrates for neurons during neural activity: a critical review of the astrocyte-neuron Lac shuttle hypothesis. J Cereb Blood Flow Metab. 2003;23:1263–1281.

13. Dienel GA, Hertz L. Glucose and lactate metabolism during brain activation. J Neurosci Res. 2001;66(5):824–838.

14. Cerdán S, Rodrigues TB, Sierra A, et al. The redox switch/redox cou- pling hypothesis. Neurochem Int. 2006;48(6–7):523–530.

15. Bolaños JP, Peuchen S, Heales SJR, Land JM, Clark JB. Nitric oxide- mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes. J Neurochem. 1994;63:910–916.

16. Almeida A, Moncada S, Bolaños JP. Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway. Nat Cell Biol. 2004;6:45–51.

17. Bigl M, Bruckner MK, Arendt T, Bigl V, Eschrich K. Activities of key glycolytic enzymes in the brains of patients with Alzheimer’s disease. J Neural Transm. 1999;106:499–511.

18. Powers WJ, Videen TO, Markham J, et al. Selective defect of in vivo glycolysis in early Huntington’s disease striatum. Proc Natl Acad Sci U S A. 2007;104:2945–2949.

19. Lehninger AL, Nelson DL. Principles of Biochemistry. New York, NY: Worth Publishers Inc; 1995.

20. Tadeusz KJ. Diagnostic difficulties in pathological fundus changes in the course of combined zygomatico-maxillary fracture. Klin Oczna. 1976;46930:341–345.

previously found in Saudi autistic patients could be supported

by considering the recent work of Gomez and Ferrer in

which they note lipoxidative damage of three enzymes linked

with glycolysis and energy metabolism in the adult human

brain.41 Gomez and Ferrer further observe that increased

oxidation of aldolase A, enolase 1, and glyceraldehydes

dehydrogenase may result in decreased activity and may

partly account for impaired metabolism and function of the

frontal lobe in Parkinson’s disease and dementia with Lewy

bodies (DLB).41

The lower PK activity found in the plasma of Saudi

autistic children compared to normal healthy control could

be correlated to the gastrointestinal disturbances that often

coexist with autism. Czub and colleagues demonstrated that

the dimeric isoform of pyruvate kinase (PK) detected in the

stool of children suffering from inflammatory bowel disease

(IBD) might serve as a potential non-invasive screening tool

for inflamed pouch mucosa.42 Enzyme immunoreactivity was

found to be significantly higher in all IBD patients than in

healthy subjects.42 Lower activity of plasma PK of autistic

children could therefore be inversely related to the higher

fecal level.

Figures 7a–d demonstrate the correlations between the

measured parameters. The positive correlations recorded

between lactate and LOX confirmed the possibility of relat-

ing these two plasmatic parameters to the etiopathology of

autism since higher lactate could explain the induced activity

of LOX. Moreover, the positive correlation between PK and

HK and the negative correlation between each of these two

enzymes and LOX could confirm the importance of lactate

and LOX as metabolic markers related to the disease.

In addition, the recorded lower activity of plasma HK and

PK could be attributed to the significantly high concentra-

tions of lead previously detected by the authors when using

the same investigated samples as the present study.43 Lead

is known to be a potent inhibitor of two sulfhydryl enzymes:

hexokinase44 and pyruvate kinase45. Moreover, Hunaiti and

Soud reported that lead may bind to and deplete glutathione

and generate reactive oxygen species.46 This finding cor-

relates to the recorded differences in glycolytic enzymes

found between autistic and control subjects, as well as the

significant depletion of reduced glutathione, and the H 2 O

2

stress previously detected in Saudi autistic children.39

Acknowledgments The authors would like to acknowledge SABIC Company,

Kingdom of Saudi Arabia for their financial support of

the present work. We also thank the families of autistic

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1. Pub Info 84:
2. Nimber of times reviewed 2: