Tryptophan metabolism and oxidative stress in patients withHuntington’s disease

N. Stoy,* G. M. Mackay,� C. M. Forrest,� J. Christofides,� M. Egerton,� T. W. Stone�and L. G. Darlington§

*Royal Hospital for Neuro-Disability, Putney, London, UK

�Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow, UK

�West Park Biochemistry Laboratories and §Department of Rheumatology, Epsom General Hospital, Epsom, Surrey, UK

Abstract

Abnormalities in the kynurenine pathway may play a role in

Huntington’s disease (HD). In this study, tryptophan depletion

and loading were used to investigate changes in blood

kynurenine pathway metabolites, as well as markers of

inflammation and oxidative stress in HD patients and healthy

controls. Results showed that the kynurenine : tryptophan

ratio was greater in HD than controls in the baseline state and

after tryptophan depletion, indicating increased indoleamine

dioxygenase activity in HD. Evidence for persistent inflam-

mation in HD was provided by elevated baseline levels of

C-reactive protein, neopterin and lipid peroxidation products

compared with controls. The kynurenate : kynurenine ratio

suggested lower kynurenine aminotransferase activity in

patients and the higher levels of kynurenine in patients at

baseline, after depletion and loading, do not result in any

differences in kynurenic acid levels, providing no supportive

evidence for a compensatory neuroprotective role for kynur-

enic acid. Quinolinic acid showed wide variations in blood

levels. The lipid peroxidation data indicate a high level of

oxidative stress in HD patients many years after disease

onset. Levels of the free radical generators 3-hydroxykynur-

enine and 3-hydroxyanthranilic acid were decreased in HD

patients, and hence did not appear to contribute to the oxi-

dative stress. It is concluded that patients with HD exhibit

abnormal handling of tryptophan metabolism and increased

oxidative stress, and that these factors could contribute to

ongoing brain dysfunction.

Keywords: Huntington’s disease, kynurenine, kynurenic

acid, oxidative stress, quinolinic acid, tryptophan.

J. Neurochem. (2005) 93, 611–623.

Huntington’s disease (HD) is an autosomal dominant disor-der characterized by neuronal loss in the striatum andneocortex. Its clinical phenotype includes motor and psychi-atric manifestations with an inexorable decline in cognitivefunction, resulting in the progressive development of choreicmovements and, in its later stages, dementia. While geneticstudies have indicated the probable involvement of a 349-kDa protein, huntingtin, bearing an abnormally expandedseries of glutamine residues close to its N-terminus, therelationship between this protein and neuronal damageremains the subject of intense investigation (Kuemmerleet al. 1999; Bates 2003).

Schwarcz and colleagues (Coyle and Schwarcz 1976;Schwarcz et al. 1984) were among the first to propose thatthe excitotoxic actions of glutamate receptor agonists couldprovide a model for neurodegenerative disorders such as HD.The discovery that quinolinic acid was an endogenousagonist at N-methyl-D-aspartate (NMDA) receptors (Stone

and Perkins 1981; Stone 2001; Stone and Darlington 2002)producing excitotoxic lesions after localized injection(Schwarcz et al. 1983) while kynurenic acid was an antag-onist (Perkins and Stone 1982) which led to hypotheses thatthese compounds, or the ratio between them, might contrib-

Received October 15, 2004; revised manuscript received December 14,2004; accepted December 14, 2004.Address correspondence and reprint requests to T. W. Stone, West

Medical Building, University of Glasgow, Glasgow G12 8QQ, UK.E-mail: t.w.stone@bio.gla.ac.ukAbbreviations used: CRP, C-reactive protein; ELISA, enzyme-linked

immunosorbent assay; ESR, erythrocyte sedimentation rate; 3-HAO, 3-hydroxyanthranilate:oxygen 3,4-oxidoreductase; HD, Huntington’s dis-ease; HPLC, high-performance liquid chromatography; IDO, indoleam-ine-2,3-dioxygenase; indole-decyclizing, indoleamine:oxygen 2,3-dioxygenase; MTBSTFA, N-methyl-N-(tert-butyldimethylsilyl)trifluoro-acetamide; NMDA, N-methyl-D-aspartate; TBDMCS, tert-butyldimethylchlorosilane; tBDMS, t-butyldimethylsilyl; TDO, tryptophan-2,3-dioxygenase.

Journal of Neurochemistry, 2005, 93, 611–623 doi:10.1111/j.1471-4159.2005.03070.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623 611

ute to the neuronal dysfunction of HD. The administration ofquinolinic acid directly into the rodent striatum reproducesmany of the neurochemical changes which have beendescribed in the HD brain (Beal et al. 1986, 1991), leadingto the widespread use of quinolinic acid lesions as anexperimental model for the disease (Schwarz et al. 1992;Popoli et al. 1994; Hughes et al. 1999). The fidelity of thequinolinic acid model is increased by localized administra-tion into the posterior putamen of non-human primates(Burns et al. 1995).

There is now strong evidence that an overexpression of theabnormal huntingtin gene contributes to the induction of HDsymptoms in humans and to the striatal characteristics of HDin mouse models (Carter et al. 1999; Sathasivam et al. 1999;Usdin et al. 1999), so it is intriguing that administration ofquinolinic acid into the striatum can induce an increasedexpression of huntingtin (Tatter et al. 1995), raising ques-tions about which comes first. Perhaps a primary toxicitycaused by kynurenine metabolites could induce huntingtin asthe cytoprotective response postulated by some authors(Kuemmerle et al. 1999; Bates 2003).

Both quinolinic acid and kynurenic acid are part of thekynurenine pathway – a major route accounting for themetabolism of over 90% of the non-protein tryptophan in mosttissues. The quantification of various kynurenine pathwaymetabolites (Fig. 1) has revealed many other examples ofpathway alterations inHD. The levels of kynurenic acid appearto be decreased in post-mortem HD brain (Beal et al. 1992;Jauch et al. 1995), with an increased ratio of kynurenine tokynurenic acid (Beal et al. 1990). Activity of the enzyme3-hydroxyanthranilic acid oxygenase (3-HAO; 3-hydroxy-anthranilate:oxygen 3,4-oxidoreductase; EC1.13.11.6), whichconverts 3-hydroxyanthranilic acid into quinolinic acid, isincreased in HD striatum (Schwarcz et al. 1988). There is alsoan increased level of 3-hydroxykynurenine in the putamen,frontal and temporal cortex of post-mortem HD which is notfound in the brains of Alzheimer’s disease patients (Pearsonand Reynolds 1992; Guidetti et al. 2000). This compoundproduces neuronal damage, largely by generating free radicalspecies such as hydrogen peroxide which induce cell apoptosisas a result of oxidative stress (Eastman and Guilarte 1989,1990; Nakagami et al. 1996; Okuda et al. 1996, 1998).

Despite the varied evidence for an alteration of kynureninepathway intermediates in the HD brain, all previous studieshave relied on static measurements of metabolite concentra-tions, often in post-mortem tissue. In contrast, this study hasexamined the kynurenine pathway activity under thedynamic conditions of tryptophan depletion and loading ina series of patients with HD compared with normal healthycontrols. We have in addition examined the levels of theinflammatory marker neopterin, the CNS inflammationmarker S100B, which is a protein released from astroglialcells, and of the lipid peroxidation products malondialdehydeand 4-hydroxynonenal as markers of oxidative stress.

Methods

Patients

Eleven patients (three men, eight women) and 15 healthy subjects

(four men, 11 women) acting as controls were recruited from the

Royal Hospital for Neuro-Disability and completed the full

experimental protocol described below. All the patients were at a

very advanced stage of HD and in a state of severe disability

requiring their permanent hospitalization in the Royal Hospital for

Neuro-disability. The numbers of subjects exclude three patients and

three controls who consumed less than 80% of the tryptophan-

depleting or -loading mixtures, or who vomited within 2 h of their

consumption. All subjects were between 18 and 65 years of age and

any patient or control taking any drug which was known to interfere

with tryptophan metabolism was excluded. Patients were recruited

after obtaining ethical permission both from the Ethical Committee

of the Epsom and St Helier University Hospitals NHS Trust and

NH

NH2

COOH

NH

O

NH2

COOH

N COOH

OH

N COOH

OH

OH

CHO

N

COOH

COOH

NH2

O

NH2

COOH

NH2

OH

NH2

COOHO

NH2

COOH

OH

OHCHOOC

COOH

NH2N COOH

N COOH

OH

tryptophan

xanthurenic acid

formylkynurenine

kynurenic acid

tryptophan-2,3-dioxygenase(TDO)

kynurenine

kynurenine aminotransferase (KAT)

quinolinic acid

3-hydroxykynurenine

3-hydroxy-anthranilic acid

kynurenine-3-hydroxylase

3-hydroxyanthranilic acid oxygenase (3HAO)

picolinicacid

glutaryl CoA

NAD

kynureninase

quinolinic acidphosphoribosyltransferase (QPRT)

indoleamine-2,3-dioxygenase(IDO)

kynurenineformamidase

8-hydroxyquinaldic acid

Fig. 1 A summary of the kynurenine pathway. Kynurenine-3-mon-

oxygenase: EC 1.14.13.9 (kynurenine-3-hydroxylase) kynurenine

2-oxoglutarate transaminase: EC 2.6.1.7 (KAT) L-kynurenine hydrol-

ase: EC 3.7.1.3 (kynureninase) tryptophan 2,3-dioxygenase: EC

1.13.11.11 (TDO) indoleamine:oxygen 2,3-dioxygenase (indole-

decyclizing): EC 1.13.11.42 (IDO) 3-hydroxyanthranilate:oxygen 3,4-

oxidoreductase: EC 1.13.11.6 (3-HAO).

612 N. Stoy et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

from the Riverside Ethical Committees of the Royal Hospital for

Neuro-Disability. Informed written consent was obtained from all

subjects or their next of kin.

All subjects were fasted overnight. Baseline blood samples were

taken through a ‘Butterfly’ cannula, and then the subjects consumed

an amino acid mixture (100 g) containing all essential amino acids,

except tryptophan. This mixture is known to induce protein

synthesis (Moja et al. 1991), leading to a depletion of plasma

tryptophan levels by 80–90% (Young et al. 1988b). Further bloodsamples were taken at 5 and 7 h after ingestion of the amino acid

mixture, i.e. when the plasma tryptophan levels were expected to

reach their lowest levels. Subjects then resumed a normal tryptophan

diet. After fasting overnight, another resting blood sample was taken

24 h after the original baseline, and a tryptophan-loading dose (6 g)

was given. Both tryptophan-depleting and -loading doses were taken

orally as fruit-flavoured solutions. Blood samples were again taken 5

and 7 h after tryptophan loading (Heuther et al. 1992). Dietary

intake throughout the study remained isocaloric and both quantita-

tively and qualitatively similar to normal for each subject. All

subjects were closely monitored by a physician throughout the

study. Blood samples were protected from light, centrifuged and

plasma and serum removed and frozen within 2 h of venesection.

Samples were maintained at ) 80�C until analysis.

Analysis of tryptophan metabolites

Samples of plasma were analysed by high-performance liquid

chromatography (HPLC), to quantify the levels of six major

components of the kynurenine pathway – tryptophan, kynurenine,

kynurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid

and xanthurenic acid. The methods have been based on the report by

Herve et al. (1996) with a number of modifications to improve peak

separation and sensitivity. During sample preparation, plasma

samples were kept on ice. To 480 lL plasma, 20 lL of internal

standard (1.2 mM 3-nitro-L-tyrosine) was added, followed by 50 lL4 M perchloric acid. Samples were vortexed for 30 s immediately

after acid addition, centrifuged at 5000 g for 10 min at 4�C, and the

supernatant collected. The precipitated proteins were resuspended in

150 lL water and 50 lL 4 M perchloric acid, the mixture vortexed

for 30 s and centrifuged at 5000 g for 10 min at 4�C. Again the

supernatant was collected. This washing and centrifugation step was

repeated and the supernatants combined. The combined supernatants

were split into two samples, one for analysis using UV/fluorescence

detection and the other for analysis using electrochemical detection.

Extracted samples were kept at ) 40�C prior to analysis. To

determine whether any metabolite was lost during the extraction

process, a spike of mixed standard solution was added to a plasma

sample before extraction. Three plasma samples were used, each

spiked at three different concentrations of mixed standard. Recov-

eries from a test batch of nine samples were > 90% for tryptophan,

kynurenine and kynurenic acid, 84% ± 7.0 (mean ± SD) for

3-hydroxykynurenine, 76% ± 8.8 (mean ± SD) for 3-hydroxyanth-

ranilic acid and 82% ± 6.9 (mean ± SD) for xanthurenic acid.

Mixed standard solutions, containing various amounts of each

standard, were prepared from frozen concentrated standard solutions.

Internal standard solution and 4 M perchloric acid were added to the

standard solutions, to ensure their compositions were comparable

with the sample extracts. Calibration curves were prepared from

chromatogram peak areas of these mixed standard solutions. For the

chromatograms at 250 nm and 365 nm, the internal standard was

included in the calibration curves and in quantifying the samples.

This compensates for any loss in recovery of the metabolites during

sample extraction. For fluorescence detection and electrochemical

detection, quantified results were later corrected for recovery, using a

recovery factor calculated from the results at 365 nm. Standard

solutions were also analysed after each block of 10 runs to check for

any drift in the retention times of the metabolites.

Defrosted sample supernatants and freshly diluted standards were

filtered through Whatman VectaSpin Micro Anopore tubes (What-

man International, Maidstone, Kent) using centrifugation at 3500 gfor 5 min at 4�C. Filtered samples and standards were kept at 4�C in

an autosampler. A volume of 100 lL was normally injected on to

the HPLC column.

Isocratic reversed-phase HPLC was performed at 37�C, using a

Waters HPLC system (Waters, Milford, MA, USA). For tryptophan,

kynurenine and kynurenic acid determination, separation was

achieved using a Kingsorb C18 column (250 · 4.6 mm ID, particle

size 5 lm, Phenomenex, Macclesfield, UK) and the detection system

included both a Waters 2487 dual wavelength UV detector (250 nm

and 365 nm) and a Waters 474 fluorescence detector, connected in

series. The mobile phase, pumped at a flow rate of 1 mL/min, was

50 mM acetic acid, 100 mM zinc acetate containing 3% acetonitrile,

based on the composition described by Herve et al. (1996). Zincacetate was included in the mobile phase as it significantly enhances

the fluorescence of kynurenic acid (Shibata 1988). Tryptophan was

determined by UV detection at a wavelength of 250 nm and

kynurenine was detected at 365 nm. Kynurenic acid was determined

by fluorescence detection (excitation 344 nm, emission 390 nm).

For 3-hydroxykynurenine, 3-hydroxyanthranilic acid and xan-

thurenic acid, separation was with a Synergi Hydro-RP 80 A column

(250 · 4.6 mm ID, particle size 4 lm; Phenomenex, Macclesfield,

UK) and detection with a Waters Concorde electrochemical detector

at an oxidation voltage of 0.65 V. The mobile phase, at a flow rate of

1 mL/min, was 50 mM phosphoric acid, 50 mM citric acid, 60 lMEDTA, 8 mM heptane sulfonic acid, 2 mM sodium chloride, with the

pH adjusted to 3.1 with potassium hydroxide, and completed by the

addition of 5% methanol.

The limits of detection using an injection volume of 100 lL and a

signal-to-noise threshold of 3, were tryptophan 20 pmol, kynurenine

5 pmol, kynurenic acid 0.2 pmol, 3-hydroxykynurenine 3 pmol,

3-hydroxyanthranilic acid 3 pmol and xanthurenic acid 2 pmol.

Levels of quinolinic acid were measured by GC/MS of the

t-butyldimethylsilyl (tBDMS) derivative, after sample cleanup using

solid phase extraction. To 1 mL plasma, 100 lL of the internal

standard (10 lM dipicolinic acid) and 2 mL 0.02 M acetate buffer

pH 2.0–2.2 was added. A Waters SepPak Vac C18 (500 mg, 3 mL)

cartridge was conditioned with 1 mL 0.5 M HC1 and successively

washed with 5 mL methanol, 2 mL water and 2 mL 0.02 M acetate

buffer pH 2.0–2.2. The sample was applied to the pre-treated

cartridge. The cartridge was then washed with 2 mL water, followed

by 2 mL hexane and dried before eluting quinolinic acid with

3 · 2 mL 2% ammonia in methanol. Then, 50 lL of 2 lg/mL

prazepam in ethanol was added to the quinolinic acid extract as an

extra recovery standard and the mixture dried. The dried extract was

mixed with 0.5 mL methanol and again dried. 20 lL of deriva-

tizing agent, N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide

(MTBSTFA) + 1% tert-butyldimethylchlorosilane (TBDMCS) was

Kynurenines and Huntington’s disease 613

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

added and the sample incubated for 2 h at 90�C, as described in the

method by Dobbie and Surtees (1997). The derivatized extract was

analysed by GC/MS using a Thermo GC8000 and MD800

quadrupole mass detector. A 1 lL sample was injected using

splitless injection at 190�C on to a 30-m · 0.2 mm RTX5 GC

column. The GC oven temperature was increased from 120�C to

230�C over 5 min followed by a rise to 260�C over 16 min. The

detector, using electron impact at 70 eV, was set to maximum voltage

monitoring ions at m/z of 338.1 and 380.1 with a dwell time of 0.3 s.

The limit of sensitivity was 10 nmol/L, with between-batch

reproducibility (based on repeat assays of spiked samples of human

serum) of < 15% at 250 nmol/L and < 10% at 800 nmol/L. The

standard curve was linear over the range 12–2500 nmol/L (r2 ¼0.999), but a quadratic curve fit was used to all batches to compensate

for any loss of linearity with GC column ageing.

Standard clinical assays

Erythrocyte sedimentation rate (ESR) was measured by a Starrsed

Automated ESR machine (Vitech Scientific, Partridge Green, UK)

which utilizes the method recommended by the International Council

for Standardization in International Council for Standardization in

Haematology 1933), based on the method of Westergren (1921).

C-reactive protein (CRP) was measured using a Behring Turbitimer.

Neopterin assay

Neopterin levels were measured in 10 lL aliquots of serum using an

immunoassay kit (Immunobiological Laboratories, Hamburg, Ger-

many). All samples were analysed in duplicate. The assay was a

competitive enzyme-linked immunosorbent assay (ELISA) where a

peroxidase-conjugated and a non-conjugated antigen competed for a

fixed number of antibody binding sites. Following incubation with

substrate solution, colour developed and the optical density was read

at 450 nm in an automated plate-reader, unknowns being quantified

by reference to known standards.

Lipid peroxidation products

An aliquot of 100 lL of serum was used for quantifying the

concentrations of the lipid peroxidation products malondialdehyde

and 4-hydroxynonenal measured using a Bioxytech LPO-586 colori-

metric assay (Biostat, Stockport, UK). The analysis involved the

reaction of N-methyl-2-phenylindole with these peroxidation productsto form a stable chromogenic indolic dimer which was estimated

spectrophotometrically at 586 nm.All sampleswere tested in duplicate.

S100B assay

S100B was measured in 50-lL aliquots of serum using a commercial

ELISA (Sangtec 100 ELISA, DiaSoria, Stillwater, MN, USA).

Standards, controls and unknown samples were incubated with a

detection antibody conjugated with horseradish peroxidase. Follow-

ing incubation in microtitre plates, the wells were washed and

incubated with a tetramethylbenzidine substrate for a further 15 min.

The enzyme reaction was stopped by the addition of tetram-

ethylbenzidine stop solution and absorbance measured at 450 nm.

Statistical analyses

A repeated measures ANOVA, followed by a Student–Newman–Keuls

multiple comparison post-test, was used when comparing

measurements from the same patients or control subjects at different

time points during the depletion or loading protocols. Comparison of

original baseline andbaseline 24 h after depletion for the samepatients

or control subjects was determined using a paired two-tailed t-test.Comparisons between patients and controls were made using an

unpaired two-tailed t-test. To overcome any masking of significant

differences due to the natural variability between human subjects,

we have also calculated the percentage changes in tryptophan

metabolites from baseline levels for individual subjects. The mean

percentage changes for control subjects and patients with HD have

then been compared at each time point using a two-tailed t-test.Results are only included in the text when they provide additional

information to that obtained from the raw data. In all cases a

significance threshold of 5% (p < 0.05) was employed.

Cross-correlations were assessed by using a correlation matrix of

all combinations of parameters, which yielded the Pearson correla-

tion coefficient and its probability level. Many of the positive

significant correlations observed were those expected on simple

biochemical grounds, such as between the levels of all the tryptophan

metabolites across the various time points of the tryptophan depletion

and loading protocols. Results are quoted only for correlations which

could not be easily predicted and which may have implications for

understanding the biology of the kynurenines.

Results

ESR and CRP

The baseline CRP level in patients with HD was5.24 ± 0.87 mg/dL (n ¼ 11), significantly higher than incontrol subjects (2.00 ± 0.73 mg/dL; n ¼ 15; p < 0.01). Thebaseline ESR level in patients was 19.7 ± 5.1 mm/h (n ¼ 11)which was not significantly different from controls(11.7 ± 2.6 mm/h, n ¼ 15). In patients with HD there wasa significant correlation between ESR and age (p < 0.001)and between ESR and CRP (p < 0.05).

Neopterin

Baseline levels of neopterin in patients with HD were4.70 ± 0.45 ng/mL (n ¼ 11), significantly higher than incontrol subjects (3.22 ± 0.21 ng/mL, n ¼ 15; p < 0.01).There was a significant correlation between age and baselineneopterin levels (p < 0.01) in HD patients and betweenneopterin and CRP (p < 0.05).

Oxidative stress

Baseline levels of the lipid peroxidation products malondial-dehyde and 4-hydroxynonenal were almost eightfold higher inpatientswithHD (6.98 ± 1.16 ng/mL,n ¼ 11) comparedwithcontrol subjects (0.88 ± 0.09 lM, n ¼ 15; p < 0.001).

S100B

There was no significant difference in baseline levels of thebrain injury marker S100B between control subjects(0.09 ± 0.01 lg/l; n ¼ 15) and patients with HD(0.13 ± 0.02 lg/l; n ¼ 11).

614 N. Stoy et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

Tryptophan metabolites

TryptophanThere was no difference between the baseline levels oftryptophan in control and patient samples (Fig. 2a). Thetryptophan-depleting mixture, as expected, produced asignificant reduction of blood tryptophan concentrations incontrols and patients (Fig. 2a). There was a significantlyhigher tryptophan level at 5 h after depletion in patientscompared with controls.

A tryptophan load raised the plasma tryptophan levels inpatients and controls compared with the baseline values(Fig. 2b), although there were no differences between thepatients and controls.

A strong correlation was observed between the levels oftryptophan and baseline ESR levels, with significance valuesranging from 0.03 to 0.005 at five of the six time points oftryptophan depletion and loading. The existence of correla-tions across such a range of times and overall tryptophanlevels suggests a strong link between tryptophan levels andthe mechanisms of erythrocyte sedimentation.

Kynurenine

Baseline kynurenine levels were significantly higher inpatients with HD compared with control subjects (Fig. 3a).This significant between-group difference was maintainedthroughout the depletion protocol (Fig. 3a; p < 0.05 to

p < 0.001). An overall, highly significant, within-groupdepletion of kynurenine in plasma occurred in both patientsand controls. It was noted, however, that kynurenine levels inthe patients continued to decline even 7 h after consuming thedepleting mixture (Fig. 3a), whereas the levels in controlsubjects had started to increase towards baseline values by 7 h.

A tryptophan load raised plasma levels in patients and con-trols, with the difference in levels between patients and con-trols remaining significant at the peak 5 h time point (Fig. 3b).

Kynurenine : tryptophan ratio (k : t)The k : t ratio provides an estimate of the activity of theenzymes indoleamine-2,3-dioxygenase (IDO; indoleam-ine:oxygen 2,3-dioxygenase (indole-decyclizing); EC1.13.11.42) and tryptophan-2,3-dioxygenase (TDO; L-tryp-tophan:oxygen 2,3-oxidoreductase (decyclizing); EC1.13.11.11). Between groups, the baseline k : t ratio washighly significantly greater in patients with HD than incontrol subjects (Fig. 4a). This difference was maintained5 h after depletion. Within groups, tryptophan depletionproduced an overall change of the ratio in controls andpatients, with a highly significant increase of the ratio at 5 hin patients and a decrease in controls.

A significant difference was again apparent betweenpatients and controls 24 h after the depletion protocol butwas lost during tryptophan loading (Fig. 4b). There was asignificant increase in k : t ratios following tryptophanloading in both patients and controls.

depletion

0

10

20

30

40

50

60

70

80

90

baseline dep +5h dep +7h dep +24h

tryp

toph

an (

µM)

Controls Patients

(a)

**+++

++++++

+++

loading

050

100

150200250300

350400450

baseline load +5h load +7h

tryp

toph

an (

µM)

(b)

+++

+++

++++++

Fig. 2 Effect of tryptophan depletion and loading on tryptophan levels

in the plasma of controls and patients with Huntington’s disease. (a)

Tryptophan concentrations at baseline level and following depletion of

tryptophan, (b) tryptophan concentrations at baseline level (24 h after

depletion) and following tryptophan loading. Values are mean ± SEM.

Between control and patients groups: ***p < 0.001, **p < 0.01,

*p < 0.05. Within-group comparison between depletion or loading and

their respective baseline values and between the original baseline and

baseline 24 h after depletion: +++p < 0.001, ++p < 0.01, +p < 0.05.

depletion

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

baseline dep +5h dep +7h dep +24h

kynu

reni

ne (

µM

)

Controls Patients

(a)

**

***

**

*+++

++

++++++

++

loading

0

20

40

60

80

100

120

140

baseline load +5h load +7h

kynu

reni

ne (

µM

)

(b)*

**

+++

+++++

+++

Fig. 3 Effect of tryptophan depletion and loading on kynurenine levels

in the plasma of controls and patients with Huntington’s disease. (a)

Kynurenine concentrations at baseline level and following depletion of

tryptophan, (b) kynurenine concentrations at baseline level (24 h after

depletion) and following tryptophan loading. Values are mean ± SEM.

Between control and patients groups: ***p < 0.001, **p < 0.01,

*p < 0.05. Within-group comparison between depletion or loading and

their respective baseline values and between the original baseline and

baseline 24 h after depletion: +++p < 0.001, ++p < 0.01, +p < 0.05.

Kynurenines and Huntington’s disease 615

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

Kynurenic acid

At baseline there was no significant difference between levelsof kynurenic acid in control subjects and patients with HD(Fig. 5a). Tryptophan depletion induced a significant fall inkynurenic acid levels in both patients and controls (ANOVA,p < 0.0008 and p < 0.0001, respectively).

Tryptophan loading produced a significant elevation in thelevels of kynurenic acid but there were no differencesbetween the patient and control groups (Fig. 5b). However,calculation of the percentage increase from baseline revealeda large percentage increase of kynurenic acid levels withtryptophan loading. This change was significantly higher inpatients (30 250% ± 7083) compared with controls(10 567% ± 2118) at 5 h after loading (p < 0.05) and at7 h (patients 6819% ± 1878, controls 2470% ± 597,p < 0.05).

Kynurenic acid : kynurenine ratio (ka : k ratio).Between groups at baseline, the ratio of kynurenic acid:kynurenine levels was significantly lower in patients than incontrols (Fig. 6a). This difference was maintained through-out the tryptophan depletion protocol.

In contrast, the difference in absolute levels betweenpatients and controls disappeared with tryptophan loading(Fig. 6b). Tryptophan loading significantly increased theka : k ratio after 5 h for both patients and controls and also at7 h for the patients.

3-Hydroxykynurenine

Levels of 3-hydroxykynurenine became undetectable duringthe tryptophan depletion protocol in control and patientgroups, so we can consider only changes on tryptophanloading. Baseline levels of 3-hydroxykynurenine at the startof the loading sequence showed a significantly lower level inpatients than in controls (Fig. 7a). Tryptophan loadingproduced a highly significant increase in the levels of3-hydroxykynurenine in both patients and controls(p < 0.0005) but, even though the mean levels remainedlower in patients during tryptophan loading, the differencebetween the means was no longer significant.

Xanthurenic acid

Levels of xanthurenic acid also became undetectable duringthe tryptophan depletion protocol in control and patientgroups. Baseline levels of xanthurenic acid at the start oftryptophan loading were significantly higher in patients thanin controls (Fig. 7b). Tryptophan loading produced a signi-ficant increase in the levels in both patients (p ¼ 0.0013) andcontrols (p ¼ 0.0005), though the difference between groupswas lost.

3-Hydroxyanthranilic acid

Levels of 3-hydroxyanthranilic acid became undetectableduring the tryptophan depletion protocol in control and

depletion

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

baseline dep +5h dep +7h dep +24h

k:t

rati

o

Controls Patients

(a)

***

***

***+++

+

+++

loading

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

baseline load +5h load +7h

k:t

rati

o

(b)

***

++++++

+++ +++

Fig. 4 Effect of tryptophan depletion and loading on the ratio of

kynurenine : tryptophan (k : t) in the plasma of controls and patients

with Huntington’s disease. (a) k : t ratio at baseline level and following

depletion of tryptophan, (b) k : t ratio at baseline level (24 h after

depletion) and following tryptophan loading. Values are mean ± SEM.

Between control and patients groups: ***p < 0.001, **p < 0.01,

*p < 0.05. Within-group comparison between depletion or loading and

their respective baseline values and between the original baseline and

baseline 24 h after depletion: +++p < 0.001, ++p < 0.01, +p < 0.05.

depletion

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

baseline dep +5h dep +7h dep +24h

kynu

reni

c ac

id (

µM)

Controls Patients

(a)

+++ +++++++

loading

0

2

4

6

8

10

12

baseline load +5h load +7h

kynu

reni

c ac

id (

µM)

(b)

+++

+++

+++

Fig. 5 Effect of tryptophan depletion and loading on kynurenic acid

levels in the plasma of controls and patients with Huntington’s disease.

(a) Kynurenic acid concentrations at baseline level and following

depletion of tryptophan, (b) kynurenic acid concentrations at baseline

level (24 h after depletion) and following tryptophan loading. Values

are mean ± SEM. Between control and patients groups: ***p < 0.001,

**p < 0.01, *p < 0.05. Within-group comparison between depletion or

loading and their respective baseline values and between the original

baseline and baseline 24 h after depletion: +++p < 0.001, ++p < 0.01,+p < 0.05.

616 N. Stoy et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

patient groups, so we can consider only the changes ontryptophan loading. Baseline levels at the start of the loadingsequence were significantly lower in patients than in controls(Fig. 7c). Tryptophan loading produced a significant increasein the levels in both patients and controls, but at 5 h theabsolute levels remained significantly lower in the patientgroup.

Quinolinic acid

The levels of quinolinic acid proved difficult to analysestatistically, in view of the large variation encountered inboth the control and HD patients. There was a difference ofaround 1000-fold between the lowest and highest baselinelevels measured in different subjects. This range did notappear to correlate with any known aspect of the subjects’medical or personal history.

Overall, there was no significant difference between thebaseline levels of quinolinic acid in the two groups (Fig. 8a).After 7 h of tryptophan depletion, quinolinic acid levels inpatients were significantly lower than in controls, althoughdepletion had no significant within group effect in eitherpatients (ANOVA, F ¼ 1.0, n.s.) or controls (F ¼ 2.89, n.s.).

Tryptophan loading increased the quinolinic acid levelssignificantly after 5 and 7 h in patients (ANOVA, F ¼ 22.96,p < 0.001) and controls (F ¼ 16.37, p < 0.01), but therewas no significant difference between patient and controlgroups.

Calculation of the percentage increase from baselinerevealed a large percentage increase of quinolinic acid levelswith tryptophan loading. This change reached11 810% ± 4479 at 5 h, and 25 426% ± 11 984 at 7 h inpatients, values significantly greater than in controls(3230% ± 1537, p < 0.05; 3358 ± 1645, p < 0.05, respec-tively).

Discussion

There are clearly drawbacks to studying peripheral changesof compounds when the primary concern is their actions inthe CNS, but we consider this study to be valuable in severalways. First, the technical and ethical considerations wouldmake it impossible to justify performing studies directly inthe CNS. Second, any genetic defect which affects thekynurenine pathway in the brain is likely to be reflected in

depletion

0.00

0.02

0.04

0.06

0.08

0.10

0.12

baseline dep +5h dep +7h dep +24h

ka:k

rat

io

Controls Patients

***** *

**++

(a)

loading

0.00

0.02

0.04

0.06

0.08

0.10

0.12

baseline load +5h load +7h

ka:k

rat

io

**

++

++

+++

(b)

Fig. 6 Effect of tryptophan depletion and loading on the ratio of ky-

nurenic acid: kynurenine (ka : k) in the plasma of controls and patients

with Huntington’s disease. (a) ka : k ratio at baseline level and fol-

lowing depletion of tryptophan, (b) ka : k ratio at baseline level (24 h

after depletion) and following tryptophan loading. Values are mean ±

SEM. Between control and patients groups: ***p < 0.001, **p < 0.01,

*p < 0.05. Within-group comparison between depletion or loading and

their respective baseline values and between the original baseline and

baseline 24 h after depletion: +++p < 0.001, ++p < 0.01, +p < 0.05.

loading

02468

1012141618

baseline load +5h load +7h

3-hy

drox

ykyn

uren

ine

(µM

)

Controls Patients

*

(a)

+++

+

+++

loading

0

2

4

6

8

10

12

14

16

baseline load +5h load +7h

xant

hure

nic

acid

(µM

)

(b)

*

+++

++

loading

0

1

2

3

baseline load +5h load +7h

3-hy

drox

yant

hran

ilic

acid

(µ M

)

(c)

*

*

+++

+++++

++

Fig. 7 Effect of tryptophan loading on 3-hydroxykynurenine, xanthur-

enic acid and 3-hydroxyanthranilic acid levels in the plasma of controls

and patients with Huntington’s disease. (a) 3-Hydroxykynurenine

concentrations at baseline level (24 h after depletion) and following

tryptophan loading, (b) xanthurenic acid concentrations at baseline

level (24 h after depletion) and following tryptophan loading, (c)

3-hydroxyanthranilic acid concentrations at baseline level (24 h after

depletion) and following tryptophan loading. Values are mean ± SEM.

Between control and patients groups: ***p < 0.001, **p < 0.01,

*p < 0.05. Within-group comparison between depletion or loading and

their respective baseline values and between the original baseline and

baseline 24 h after depletion: +++p < 0.001, ++p < 0.01, +p < 0.05.

Kynurenines and Huntington’s disease 617

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

changed metabolism throughout the body, both because thesame enzymes outside the brain may be affected and becauseof the passage of metabolites from CNS to the blood and viceversa. In fact the levels of kynurenine metabolites in the CNSare substantially influenced by peripheral levels, especially astryptophan and kynurenine penetrate readily across theblood–brain barrier using the large neutral amino acidtransporter (Fukui et al. 1991; Miller et al. 1992; Vecseiet al. 1992; Jauch et al. 1993) and induce increased produc-tion of downstream metabolites (Jauch et al. 1993). This isparticularly likely to be a significant factor in neurodegen-erative disorders such as HD as any accompanying inflam-matory activity will result in the infiltration into the CNS ofactivated macrophages with enhanced kynurenine pathwaymetabolism promoted by the activation of IDO activity(Heyes et al. 1992b; Espey et al. 1997).

Kynurenines

There are two primary hypotheses for a role of tryptophanmetabolites in HD. One is based on the ability of kynureninesto modulate activity of the NMDA-sensitive glutamatereceptors, while the other is based on the ability of3-hydroxykynurenine and 3-hydroxyanthranilic acid tomodulate the generation of reactive oxygen species and,thus, the production of oxidative stress.

A crucial role for NMDA receptors in the neuronaldysfunction and loss which occurs in HD has been postulatedsince the concept of a relationship between excitotoxicity andneurodegenerative disease was proposed by Coyle andSchwarcz (1976) and the excitatory and neurotoxic actionsof quinolinic acid as an endogenous agonist at NMDAreceptors was described (Stone and Perkins 1981; Schwarczet al. 1983). In post-mortem HD brain, Young et al. (1988a)detected a loss of NMDA receptors, consistent with anincreased loss of cells bearing these receptors in response toan excitotoxic process mediated by glutamate or quinolinicacid. The huntingtin protein increases expression of the NR1/NR2B subunit combinations in the NMDA receptors foundon the medium spiny neostriatal neurons which are mostvulnerable to loss in HD. This effect could account for theincreased neuronal sensitivity to NMDA reported in a mousemodel (Cepeda et al. 2001), and the increased sensitivity toNMDA or quinolinic acid excitation and toxicity (Chen et al.1999; Zeron et al. 2002).

While there is no apparent difference between the restinglevels of tryptophan in patients with HD and controls in thisstudy it is clear that there is a far greater conversion oftryptophan to kynurenine in patients because the absolutebaseline levels of kynurenine are more than double those incontrols, i.e. the kynurenine pathway appears to be in a stateof enhanced activation in HD. It is of interest, however, thatkynurenine levels fall to a much greater extent at 5 h oftryptophan depletion in controls than in patients. Thissuggests that the relatively greater activity of TDO/IDO inpatients is increased even further in this situation. The raisedlevels of kynurenine in HD are believed to demonstrate a realeffect as they are observed at five of the six time pointsthroughout the depletion and loading protocols. Indeed,calculation of the kynurenine : tryptophan (k : t) ratiosreveals that at the time of maximum tryptophan depletionthe ratio falls in controls but is increased in patients.Together, these results suggest that the activity of TDO and/or IDO is greater in patients than controls, and that activity isnot depressed as much by lowered tryptophan availability asit is in controls.

This difference may be due more to the availability ofcofactors or competing substrates than to a difference in theenzymes themselves, as the difference in activity is lostfollowing a tryptophan load, when the enzymes should bemaximally active and competition for alternative substrateswill be heavily in favour of tryptophan. This in turn maysuggest that IDO contributes relatively more to the metabo-lism of tryptophan in patients, as this enzyme acts on a widerrange of substrates than does the more selective TDO. Agreater contribution by IDO would also be entirely consistentwith the greater neopterin levels and oxidative stressproducts in patients, as IDO is present in microglia andmacrophages and other immune competent cells.

depletion

0

1

2

3

4

5

baseline dep +5h dep +7h dep +24h

quin

olin

ic a

cid

(µM

)

Controls Patients

***

(a)

loading

0

2

4

6

8

10

12

14

baseline load +5h load +7h

quin

olin

ic a

cid

(µM

)

(b)

++++

++

+

Fig. 8 Effect of tryptophan depletion and loading on quinolinic acid

levels in the plasma of controls and patients with Huntington’s disease.

(a) Quinolinic acid concentrations at baseline level and following

depletion of tryptophan, (b) quinolinic acid concentrations at baseline

level (24 h after depletion) and following tryptophan loading. Values

are mean ± SEM. Between control and patients groups: ***p < 0.001,

**p < 0.01, *p < 0.05. Within-group comparison between depletion or

loading and their respective baseline values and between the original

baseline and baseline 24 h after depletion: +++p < 0.001, ++p < 0.01,+p < 0.05.

618 N. Stoy et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

In normal controls, the k : t ratio falls on depletion andrises on loading as might be expected as the pathways have agreater or lesser degree of activation, respectively. In HDpatients, however, the k : t ratio rises both on tryptophandepletion and loading and in this study was always higherthan in controls at all points in the depletion and loadingprogramme, suggesting that IDO/TDO activity is alwaysgreater in HD than in controls, perhaps fuelled by superoxidewhich acts as a substrate for IDO, or under the persistentinfluence of some factor released from the damagedneurones.

Although kynurenic acid is an antagonist at all glutamateionotropic receptors, it has its greatest activity at thecoagonist allosteric glycine site (the glyB receptor) on theNMDA receptor (Birch et al. 1988). It is able therefore toblock neurotransmission and brain damage induced byexcitotoxins, brain oedema, hypoxia and ischaemia (seeStone 2001). In the present study, the baseline and trypto-phan depleted levels of kynurenic acid were no differentbetween patients and controls. While the kynurenicacid : kynurenine ratio (ka : k) would be consistent with asubstantially reduced kynurenine aminotransferase (KAT;kynurenine 2-oxoglutarate transaminase, EC 2.6.1.7) activityin patients at baseline and on tryptophan depletion, such aconclusion must be qualified by the fact that kynurenic acidis excreted via active transport in the kidney, and KATactivity depends on the simultaneous concentrations ofalternative substrates and modulators such as aspartate,a-aminoadipate and oxoglutarate. It is not known whetherthese factors were different in the patient population studied.The overall picture is nevertheless one of increased IDO/TDO activity driving the kynurenine pathway towardsquinolinic acid in the presence of lower concentrations ofthe protective kynurenic acid.

It may be important that on tryptophan loading thedifferences between HD patients and controls is lost, raisingthe possibility that loading the pathway might be protectivein driving the balance in the pathway towards kynurenic acidand away from quinolinic acid. Interestingly, however, thereis a case report by Pascoe (1993) who claimed thestabilization of a HD patient by a low tryptophan diet. Thisresult might indicate that a lower generation of quinolinicacid is more beneficial than raising the kynurenic acid : qui-nolinic acid ratio.

The earliest measurement of kynurenic acid levels in thepost-mortem brains of patients with HD indicated anincrease (Connick et al. 1989), whereas later investigationsin a larger sample of post-mortem brains (Beal et al. 1992)and in the CSF (Heyes et al. 1992a) indicated a decrease,together with an increased ratio of kynurenine to kynurenicacid (Beal et al. 1990). However, a recent study of earlystage HD brains and an early stage HD mouse modeltransgenic for full-length mutant huntingtin has againreported an increase of kynurenic acid levels (Guidetti

et al. 2000), although accompanied by a even greaterincrease of 3-hydroxykynurenine. The present data do notdirectly support either of these alternatives, although ourfinding of a substantially reduced ka : k ratio is entirelyconsistent with the report by Jauch et al. (1995) that KATactivity was less in the HD post-mortem caudate nucleusthan control brains. One very interesting possibility arisesfrom the known ability of blood kynurenine to pass into theCNS and be converted into kynurenic acid (Miller et al.1992; Vecsei et al. 1992; Jauch et al. 1993). Because bloodkynurenine levels are very significantly higher in patientsthan controls, it may lead to a raised level of kynurenic acidin the brain, as reported by Connick et al. (1989) in humanbrain, and in the animal model (Fukui et al. 1991; Milleret al. 1992; Vecsei et al. 1992; Jauch et al. 1993; Guidettiet al. 2000).

Although the large variability of quinolinic acid levels inthe present cohort of subjects makes interpretation difficult,the absence of a clear difference between patients andcontrols is consistent with earlier reports in post-mortembrain (Reynolds et al. 1988; Beal et al. 1990; Reynolds andPearson 1993). It has been pointed out by Poston et al.(1991) that the low Km of 3-HAO and the presence of3-HAO in astrocytes means that increased baseline kynur-enine levels may not be reflected in a proportionate rise inbaseline quinolinic acid levels, and this is borne out by thepresent results. Activity of 3-HAO is said to be increased inHD striatum (Schwarcz et al. 1988), and this may accountfor the normal baseline level of quinolinic acid in the presentstudy despite a decreased concentration of 3-hydroxyanthra-nilic acid.

Oxidative stress

There is growing evidence for a degree of oxidative stress inHD which could result from the activation of NMDAreceptors [which are known to induce nitric oxide synthase(iNOS; EC 1.14.13.39) and the generation of reactive oxygenspecies] or by some other method independent of thereceptors. In at least one animal model for HD, a significantincrease in lipid peroxidation has been reported whichparallels the neurological phenotype (Perez-Severiano et al.2000; Bogdanov et al. 2001).

An increased level of 3-hydroxykynurenine has beendescribed in the putamen, frontal and temporal cortex in HDwhich is not found in the brains of Alzheimer’s diseasepatients (Pearson and Reynolds 1992; Guidetti et al. 2000).This compound produces neuronal damage, largely bygenerating free radical species such as hydrogen peroxidewhich induce cell apoptosis as a result of oxidative stress(Eastman and Guilarte 1989, 1990; Nakagami et al. 1996;Okuda et al. 1996, 1998).

The present results are certainly consistent with elevatedoxidative stress as reflected in the significantly raised levelsof lipid peroxidation products. As ESR correlated positively

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� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

with age only in the HD patients and not in controls it may bethat the inflammation shown in these patients, i.e. increasedneopterin and lipid peroxidation products are not solely theeffects of ageing but reflect an inflammatory process in thebrains of these patients resulting from the disease process andpossibly a response to proteins or other substances releasedfrom the damaged brain as the disease progresses with time.ESR correlated positively with CRP (p < 0.05) which isgenerally accepted to be the more reliable measurement ofdisease activity.

IDO activity is known to be stimulated by superoxide,which acts as a co-substrate for the enzyme. It is possiblethat oxidative stress leading to superoxide production couldresult in a secondary activation of the kynurenine pathway,generating the raised kynurenine level and k : t ratio.Alternatively, a primary increase of kynurenine pathwayactivity could increase oxidative stress as a result of raisedlevels of metabolites such as 3-hydroxykynurenine and3-hydroxyanthranilic acid which can generate reactiveoxygen species. Because resting levels of these lattermetabolites are shown here to be lower in patients than incontrols, the former explanation is more likely to becorrect, although it should be emphasized that, as a resultof the peripheral generation and metabolism of thesecompounds, our measurements of these compounds inblood may reflect the central situation to only a limitedextend, with the possibility that their levels could be raisedwithin the CNS.

There are related observations to suggest an impairment ofcellular energy metabolism in animal models and HDpatients (Beal 1992; Browne et al. 1999) which could resultfrom the binding of huntingtin to key enzymes such asglyceraldehyde-3-phosphate dehydrogenase (D-glyceralde-hyde-3-phosphate:NADP+ oxidoreductase, EC 1.2.1.9;Burke et al. 1996). These changes include defects inmitochondrial function (Tabrizi et al. 2000) with increasedNOS activity, and could contribute to the increased sensitiv-ity of NMDA receptors in HD (Beal 1992) as well as thegeneration of free radical species both directly and as a resultof NMDA receptor activation.

Neopterin

The increased level of neopterin in our HD patients whencompared with controls is consistent with evidence for aninflammatory process in neurodegenerative disorders suchas Parkinson’s disease (Widner et al. 2002) and Alzheimer’sdisease, and may reflect an inflammatory reaction to cellulardamage and removal occurring within the CNS. It is stillnot clear whether the peripheral immune activation is aprimary response, and the activated peripheral macrophageswhich are known to penetrate into the CNS are thenrecruited to the sites of cell damage, or whether the primaryresponse is activation of microglia by cellular damagewithin the CNS. Activated microglia are present in HD

brain from an early stage in proximity to the most affectedareas (Sapp 2001) and iNOS has been found in (activated)microglia adjacent to degenerating neurones (Tabrizi et al.2000). Macrophages derived from peripheral blood mono-cytes are well known to be recruited into regions of CNSinflammation and necrosis but their contribution to HDpathogenesis is unknown.

Raised levels of neopterin are often associated withincreased activity of reactive oxygen species and low levelsof antioxidants (Murr et al. 2002), an observation which issupported by our present finding of similarly elevated levelsof neopterin and lipid peroxidation products in HD patients.Activated microglia are present in HD brain from an earlystage in proximity to the most affected areas (Sapp 2001) andiNOS has been found in (activated) microglia adjacent todegenerating neurones (Tabrizi et al. 2000). Macrophagesderived from peripheral monocytes are well known to berecruited into regions of CNS but their contribution to HDpathogenesis is unknown.

The raised level of neopterin in patients with HD isconsistent with the similar finding from Leblhuber et al.(1998) who also noted a correlation with lower tryptophanlevels and with poor survival of patients with HD.

CRP

Elevated levels of CRP are associated with the immuneresponse and disease activity (Gershov et al. 2000). Raisedlevels have been correlated with the inflammatory eventsaccompanying heart disease and atherosclerosis, with nocorrelation with antibodies to infectious agents (De Backeret al. 2002; Heilbronn and Clifton 2002). The raised levels ofCRP found here therefore in the absence of changes of ESRand haemoglobin, could be an indicator of generalizedinflammatory response to the destruction of brain cells andhuntingtin-related damage elsewhere, as for example instriated muscle, though the specific contribution of CRP tothe inflammatory response remains uncertain. This inflam-matory response could result in the activation of thekynurenine pathway with potentially yet more inflammation,i.e. a self-perpetuating cycle.

S100

This is one of the first reports of the measurement of S100Bin patients with HD. The protein S100B is released fromastroglial cells and is a biochemical marker for diagnosis andmonitoring of brain damage. S100B is elevated in patientswith Alzheimer’s disease and Down’s syndrome (Griffinet al. 1989), and is increased following ischaemic stroke(Buttner et al. 1997; Wunderlich et al. 1999). The normallevels in blood found here are consistent with evidence thatS100B is associated with the earliest stages of neuronaldamage, and that levels decline within a few days of an acuteinsult. The absence of a difference from controls thereforehelps to rule out the existence of acute cerebral damage

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� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 611–623

superimposed upon the chronic neurodegeneration. Theresults also suggest that S100B has no value as a markerof neurodegeneration in later stages of HD.

In summary, this study has demonstrated clear differences inthe levels of oxidative stress and the kinetics of the kynureninepathway in patients with HD compared with controls.Tryptophan depletion or loading gives rise to differentialeffects in patients and controls both in terms of the absoluteamounts of metabolites produced, and in terms of the timecourse of those changes. These differences could reflectgenetic differences in the various enzymes of the pathway,differences in the concentrations or availability of cofactors orcompeting substrates for the enzymes, or differences in theinteractions between the kynurenine pathway and metabolicpathways which compete with key metabolites (Fig. 1). Theseperipheral results may not accurately reflect concentrations inthe CNS, but they are consistent with the general concept thatabnormalities of tryptophan metabolism along the kynureninepathway could contribute to neuronal dysfunction and damagein HD. The data also strongly reinforce the view that a highlevel of oxidative stress is present in HD patients many yearsafter the onset of the disease.

Acknowledgements

The authors wish to acknowledge Ms Rosalind B. McMillan for

technical assistance in Glasgow, and the invaluable help of

Mrs Helen Finch, MSc SRD (Dietician) and of Dr M. Khanderia

(Chief Pharmacist) at The Royal Hospital for Neuro-Disability,

London. They also wish to thank The Peacock Trust and the Royal

Hospital for Neuro-Disability for their support.

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