author. Dr Hugh Rickards
email: hugh.rickards@bsmhft.nhs.uk

Jenny Keylock

Marleen Van Walsem

Dr Erik Van Duijn

Dr Sheila Simpson

Dr Ferdinando Squitieri

Bernhard Landwehrmeyer

MSc Md * Jenny Marleen Keylock

Ms. Marleen Erik Van Walsem

MD Van Duijn

A Sheila

M D Simpson

MD Bernhard Ferdinando Squitieri

MD. Landwehrmeyer

Ms Jenny Keylock

Prof Bernhard Landwehermeyer

factor analysis of behavoiural symptoms in Huntington's Disease

Keywords: Huntington's disease, factor analysis, behaviour, mental disorder

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Principal components factor analysis revealed 3 clusters of symptoms, reflecting apathy, depression and irritability. The apathy factor was closely related to the duration of illness, being a stronger feature later in the illness. A further paper by Kingma et al 6 using the PBA on 152 HD mutation carriers also showed a 3 factor solution; apathy, depression and irritability.

The European Huntington's Disease Network (EHDN) REGISTRY study (www.eurohd.net/html/registry) has provided a unique opportunity to re-examine psychiatric symptoms in the setting of HD. This study has recruited around 4000 people with HD and participants are followed up at yearly intervals.

The aim of our study was to perform a factor analysis on all the completed UHDRS behavioural assessments performed across Europe since the inception of the REGISTRY study. The total number of fully completed assessments at the time of writing was 1690. Elucidation of specific behavioural or psychiatric factors may lead to the identification of specific phenotypes within HD, which in turn could lead to greater insight into aetiology and aid with treatment.

Method.

Data were made available from the EHDN REGISTRY database following approval from the Scientific and Bioethical Advisory committee and the Executive committee of the EHDN.

UHDRS behaviour scores were obtained for 1803 assessments on individual participants. The UHDRS behaviour section is made up of 11 individual items, each scored on the dimensions of frequency and severity.

The frequency (score 0-4 points) and severity (score 0-4 points) of each item were multiplied to give a single score for each item. The item scores are then added together to give the total behaviour score.

Principal components analysis was employed, with the "frequency multiplied by severity" score for each item used as a factor. Data on all the factors were analysed using SPSS v.16. A Varimax rotation was utilised with Kaiser Normalisation. Due to missing data, 113 cases were excluded to give a final N of 1690. Full ethical approval was obtained for this study.

Results. Discussion.

This factor analysis builds on the findings of Craufurd et al These data further support the existence of specific clusters of psychiatric symptoms in HD, which may be related discretely to affect, executive function, aggression/irritability and perception. More research is needed to assess the validity of these categories, as well as their stability over time, including in the pre-manifest period. Further work is needed to develop valid rating scales that measure change in these factors as a result of treatment.

Conclusion.

Distinct clusters of behavioural symptoms are emerging in HD. These relate in general to depression, executive function, irritability and psychosis. Further studies should aim to tie in these factors with specific endophenotypes in order to establish clear clinico-pathological correlation.

2 . 3 .

 23 Introduction Huntington's disease (HD) is a progressive neuropsychiatric disorder consisting of motor, cognitive, behavioural, affective and perceptual changes. Psychiatric problems in HD are common and lead to significant impairment in the Quality of Life of people with HD 1, The Behaviour section of the Unified Huntington's Disease Rating Scale (UHDRS) was designed to record a range of psychiatric phenomena in people with HD There are few studies analysing the relationship between different psychiatric phenomena in HD. Marder et al 4 performed a factor analysis on 960 UHDRS assessments as part of the Huntington's Study Group (HSG) project. They isolated 15 factors, including "depression/anxiety", "suicidal thoughts", "aggressivity" and "obsessive/compulsive". There were a number of smaller factors including hallucinations in different modalities, something not routinely enquired about in current UHDRS assessment. Craufurd et al 5 used factor analysis to examine data on 78 patients with HD who had undergone the Problem Behaviours Assessment in HD (PBA-HD).

  old version of the UHDRS Behaviour assessment. All the studies have shown distinct factors for depression, executive functions and irritability.Both studies using the UHDRS behaviour section had separate factors for psychosis.

Table 1 .

 1 Factor analysis of behavioural symptoms in Huntington'sDisease. N=1690

	Extraction method: Principal Components Analysis
	Rotation method: Varimax with Kaiser normalisation
	Data included in bold if factor > 0.4
	As seen in Table 1, four factors achieved a loading of over 0.4. These factors accounted with 63.8%
	of variance in the total score. The four factors were;
	1.	Depression (depressed mood, low self-esteem, suicidal ideation, anxiety)

Acknowledgements.

The authors would like to acknowledge the behavioural phenotype working group of the EHDN (www.euro-hd.net/html/network/groups/behavioural) and CHDI Foundation, Inc.

The list of contributors to the data set is given below (Appendix 1).

Competing Interests

This study received no sponsorship and was led by the investigators. Dr Rickards has sat on an advisory board for MEDA pharmaceuticals. He has been paid honoraria for work for Cambridge Laboratories. Dr. Simpson received an honorarium from Cambridge Pharmaceuticals for consultancy. Jenny Keylock reports no disclosures Marleen van Walsem reports no disclosures Dr van Duijn reports no disclosures Dr. Squitieri reports no disclosures Prof. Landwehrmeyer reports no disclosures

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Professor Mamede De Carvalho
email: mamedemg@mail.telepac.pt

MD FRCP FRCPath Michael Swash

Fasciculation-cramp syndrome preceding anterior horn cell disease: an intermediate syndrome?

Keywords: after-discharges, amyotrophic lateral sclerosis, crampfasciculation syndrome, fasciculations, myokymia

Cramp-fasciculation syndrome (cramp-FS) is an ill-defined condition with uncertain clinical limits. We studied a 55-year-old man with progressively more severe, widespread fasciculations and cramps during a six-year period. Mild progressive lower motor neuron loss, shown by MUNE and multiMUP analysis, developed during the four years after onset, which stabilized during a further 2-year follow-up. Cramp-FS is generally a benign syndrome, probably with several causations. Our patient developed a limited form of anterior horn cell degeneration perhaps representing a syndrome transitional with amyotrophic lateral sclerosis.

Cramp-FS merits more detailed study.

INTRODUCTION

The cramp-fasciculation syndrome (cramp-FS), occurring without weakness or atrophy, is generally regarded as a benign, non-progressive disorder. [START_REF] Blexrud | Long-term follow-up of 121 patients with benign fasciculations[END_REF] However, the clinical limits of this syndrome are ill-defined. Indeed, fasciculations occur frequently in normal subjects. [START_REF] Layzer | The origin of muscle fasciculations and cramps[END_REF][START_REF] Van Der Heijden | fasciculation potentials in foot and leg muscles of healthy young adults[END_REF] Cramp-FS probably represents hyperexcitability in lower motor neurons, causing after-discharges and myokymia-like spontaneous activity on EMG. [START_REF] Denny-Brown | Myokymia and the benign fasciculation of muscular cramps[END_REF][START_REF] Vincent | Understanding neuromyotonia[END_REF][START_REF] Tahmoush | Crampfasciculation syndrome: a treatable hyperexcitability peripheral nerve disorder[END_REF][START_REF] Warmolts | impulse-induced repetitive discharges in motor nerves in peripheral neuropathy[END_REF][START_REF] Parisi | After-discharge activity in neuropathic patients with frequent muscle cramps[END_REF][START_REF] Harrison | Accuracy of repetitive nerve stimulation for diagnosis of the cramp-fasciculation syndrome[END_REF] Since patients with amyotrophic lateral sclerosis (ALS) often describe fasciculation and cramp before progressive weakness develops, [START_REF] De Carvalho | Cramps, muscle pain, and fasciculations: not always benign[END_REF][START_REF] Fleet | From benign fasciculations and cramps to motor neuron disease[END_REF] these symptoms not infrequently raise fears of ALS. We report an atypical patient whose clinical and EMG features bridge the benign condition of cramp-FS and ALS, as defined by anterior horn cell disease without upper motor neuron features.

METHODS

We have followed convention in describing fasciculation potentials (FPs) as spontaneous, non-volitional, potentials causing a muscle twitch with an EMG morphology similar to the motor unit potential, typically with shape variability, in the absence of myokymia and after-discharges. [START_REF] Layzer | The origin of muscle fasciculations and cramps[END_REF][START_REF] Buchtal | Spontaneous electrical activity: an overview[END_REF] Cramps were defined as painful sudden muscle contractions with electrical activity on EMG, induced by muscle contraction and relieved by muscle stretching.

Motor unit number (MUNE) in hand muscles was calculated by the incremental technique, as described elsewhere. [START_REF] Carvalho | Quantitating progression in ALS[END_REF] Conventional MultiMUP analysis was used to study motor unit potentials and FPs in concentric needle EMG recordings.

CASE REPORT

A 55-year-old man presented with widespread fasciculations, involving upper and lower limb muscles and chest muscles, but not cranial muscles, and with prominent subjective fatigue. Fasciculations were present at rest, and not apparently triggered by exercise. He was not taking medication. There was no relevant personal or family history. Clinical examination and laboratory work-up were normal. EMG studies (not shown in Table 1) performed shortly after symptom onset (MdeC) showed no abnormality other than FPs of normal morphology in biceps and gastrocnemius muscles bilaterally. In these muscles, and in first dorsal interosseous (FDI), tibialis anterior and vastus medialis, motor units were stable and of normal morphology without fibrillations or positive sharp waves (fibs-sw). Nerve conduction studies were normal. He was anxious and complained of mild headache; a brain MR scan was normal. A diagnosis of benign fasciculations was made and an anxiolytic was recommended.

Two years later he developed cramps, provoked by mild muscular contraction, affecting trunk, abdominal, neck, and upper and lower limb muscles. Cramps were also present at night. In addition, fasciculations seemed more frequent. He consulted a neurosurgeon who arranged cervical, dorsal and lumbosacral MR imaging, which was unremarkable.

Laboratory tests, including ESR, electrolytes, creatinine clearance, serum calcium and phosphorous, magnesium, protein immunoelectrophoresis, and thyroid and parathyroid hormone levels were normal. Antiganglioside antibodies were negative. He declined CSF examination. He was referred to us.

He was anxious but unaware of a possible diagnosis of amyotrophic lateral sclerosis (ALS). Fasciculations were noted in arms and legs, mainly in proximal muscles. Voluntary contraction of thigh, calf, forearm, abdominal and paraspinal muscles induced prominent cramps. Cranial muscles were normal. Strength was normal in all muscle groups and there was no atrophy. Tendon reflexes were normal, plantar responses were flexor and sensory examination was normal. Respiratory function tests were normal. On neurophysiological examination nerve conduction studies and skin sympathetic responses were normal. Repetitive stimulation of the right posterior tibial nerve at 2Hz and 5Hz did not induce sustained after-discharges of the abductor hallucis brevis muscle, [START_REF] Tahmoush | Crampfasciculation syndrome: a treatable hyperexcitability peripheral nerve disorder[END_REF] indicating no increased motor nerve excitability. There was no conduction block and Fwave studies were normal. Concentric needle EMG sampling (Table 1; investigation A) revealed FPs in both biceps, tibialis anterior and vastus medialis muscles and in the left FDI, with large polyphasic motor units in left biceps and FDI and both tibialis anterior muscles, confirmed by multiMUP analysis. FPs showed complex morphology and moderate instability but there were no fibs-sw. Quantitative EMG analysis of the FPs revealed large potentials of long duration. There was no other spontaneous motor unit activity.

Over the next 4 years abundant fasciculations and frequent cramps, often elicited by mild muscular effort, with fatigue, continued to be troublesome. Muscle power and deep tendon reflexes remained normal.

Repeated clinical and neurophysiological evaluations were performed at 6 month intervals during this period (Table 1). Overall, the EMG results showed that the lower motor neuron pool, as shown by MUNE studies, decreased between the second and fourth years after presentation, but remained constant during the next two years. Chronic partial denervation developed during this time, although fibs-sw were not detected. A recent gadolinium-enhanced MRI of the brain and spinal cord requested by another neurologist showed no abnormality.

DISCUSSION

The causation of cramp-FS is not understood; it may represent a spectrum of disorders. [START_REF] Layzer | The origin of muscle fasciculations and cramps[END_REF] Early descriptions illustrate this. For example, Denny-Brown and Foley 4 described two patients with myokymia and cramps who perspired profusely, and had neuromyotonic discharges on needle EMG. They noticed that the FPs cancelled out a series of recruited motor units indicating a peripheral origin for FPs in the distal axonal arborisation. In addition, mild exercise during circulatory occlusion was effective in precipitating fasciculation and cramps in predisposed subjects.

They suggested that myokymia had a proximal origin and that it could be induced by release from limb ischaemia. Neuromyotonia is characterised by recurrent axonal discharges causing repetitive motor unit potentials in EMG recordings; it has subsequently been recognised as an auto-immune disorder due to antibodies to voltage-gated potassium channels at motor end-plates. [START_REF] Vincent | Understanding neuromyotonia[END_REF] In 1978 Hudson and co-workers [START_REF] Hudson | The muscular pain-fasciculation syndrome[END_REF] described five patients with fasciculations, muscle pain and cramps. Four showed signs of active denervation in lower limb muscles suggesting a diagnosis of peripheral neuropathy, anterior horn cell disease or root lesion. In another report [START_REF] Coers | Neurogenic benign fasciculations, pseudomyotonia and pseudotetany[END_REF] two patients with neuropathy associated with typical features of neuromyotonia were described as "neurogenic benign fasciculations", a terminology that invited confusion. In 1991 Tahmoush and co-workers [START_REF] Tahmoush | Crampfasciculation syndrome: a treatable hyperexcitability peripheral nerve disorder[END_REF] defined cramp-FS as characterized by myalgia, cramps, stiffness, exercise intolerance and increased peripheral nerve excitability. Their nine patients had fasciculations or myokymia, mostly in calf muscles. However, this description most likely represented neuromyotonia, which is associated with stiffness (60%) cramps (12%), weakness (12%) and muscle twitching (4%). [START_REF] Jameson | Idiopathic generalized myokymia[END_REF] Tahmoush et al [START_REF] Tahmoush | Crampfasciculation syndrome: a treatable hyperexcitability peripheral nerve disorder[END_REF] did not describe their EMG findings in detail, but repetitive nerve stimulation at 5 Hz in lower limb nerves induced after-discharges following the M-waves. After-discharges are often noted during nerve conduction studies of patients with peripheral neuropathies, as described by Warmolts and Mendell in 1980, [START_REF] Warmolts | impulse-induced repetitive discharges in motor nerves in peripheral neuropathy[END_REF] either immediately following M-waves (peripheral hyperactivity) or after the Fwave (lower motor neuron hyperexcitability). [START_REF] Parisi | After-discharge activity in neuropathic patients with frequent muscle cramps[END_REF] Baldissera and coauthors [START_REF] Baldissera | Motor Neuron "bistability". A pathogenic mechanism for cramps and myokymia[END_REF] presented evidence that myokymia and cramps may occur as manifestations of lower motor neuron bistability, an anomalous condition in which the motor axonal cell membrane has two equilibrium levels, a lower level near the resting membrane potential and a higher one above the threshold for generation of self-sustained rhythmic firing.

Fasciculation potentials were defined by Buchthal 12 as brief, small muscle twitches, representing motor units or parts of motor units. A FP is a spontaneous action potential similar in size and duration to a motor unit potential. In ALS or other neurogenic disorders, FPs show differing morphology in consecutive twitches, and may be more or less complex. In normal human subjects, they can be induced by cholinergic drugs, and they are abolished by curare in doses insufficient to block neuromuscular transmission, suggesting an effect on motor nerve terminals. Cramps may result from regional spread of successive fasciculations of distal origin; therefore, they also originate in the intramuscular nerve terminals, 2 as well as proximally from motor nerve cells. [START_REF] Baldissera | Motor Neuron "bistability". A pathogenic mechanism for cramps and myokymia[END_REF] Since many patients with ALS report that their first symptom was fasciculation, either focally in a group of muscles or more diffusely, there is often concern at presentation as to whether cramp-FS might represent the onset of ALS. Indeed, we have reported such a case. [START_REF] De Carvalho | Cramps, muscle pain, and fasciculations: not always benign[END_REF] In ALS, however, other features such as muscle fatigue, weakness, muscle atrophy and upper motor neuron signs, are necessary to make the diagnosis, and in cramp-FS follow-up generally excludes a diagnosis of ALS. Moreover, in ALS, FPs are complex, and polyphasic, with striking variability in shape, due to jitter and blocking within the potential. [START_REF] Carvalho | Electrodiagnostic criteria for diagnosis of ALS: Consensus of an International Symposium sponsored by IFCN, December 3-5 2006, Awaji-shima, Japan[END_REF] In cramp-FS, FPs are of simple morphology, without associated reinnervated, neurogenic change. [START_REF] Blexrud | Long-term follow-up of 121 patients with benign fasciculations[END_REF] In our patient an EMG soon after the onset of symptoms disclosed no abnormality apart from FPs of benign morphology. Two years later reinvestigation revealed neurogenic changes in limb muscles, raising a suspicion of anterior horn cell disease, although muscle strength was normal, and there was no muscle atrophy or upper motor neuron sign.

Six years after onset of cramp-FS, there were EMG features of chronic partial denervation, with increased complexity and moderate instability of motor unit potentials and FPs. After-discharges and myokymia were not found, [START_REF] Tahmoush | Crampfasciculation syndrome: a treatable hyperexcitability peripheral nerve disorder[END_REF] and there were no features suggestive of peripheral neuropathy.

We concluded that this was an acquired, non-progressive anterior horn cell disorder, that represented a benign variant of the progressive muscular atrophy form of motor neuron disease The somewhat ill-defined clinical syndrome of cramp-FS without myokymia or after-discharges deserves more attention. It is likely to subsume a number of different disorders ranging from a benign condition(s) to the onset of ALS. Our observation of transition from cramp-FS to anterior horn cell disorder, entering a period of stability after a phase of initial progression, has implications for understanding the onset of ALS, since ALS is often heralded by fasciculations, representing spontaneous motor unit activity. In particular, it contributes to understanding ALS patients who stabilize after an initial progressive phase. [START_REF] Tucker | Subacute, reversible motor neuron disease[END_REF] In contrast to our patient in an Australian family with a SOD1related familial form of ALS, a remarkably abrupt loss of motor units was noted some weeks prior to the onset of detectable muscle weakness. [START_REF] Aggarwal | Detection of preclinical motor neuron loss in SOD1 mutation carriers using motor unit number estimation[END_REF] The wide range of severity and progression of lower motor unit involvement in the early stages of ALS syndromes is a characteristic but ill-understood feature of the disease. Indeed, understanding the dynamics of ALS onset and of the different syndromes that are currently included in the classification of the common forms of motor neuron diseases is a fundamental question that at present remains unresolved. There was a reduction in motor unit number between the 2-year and the 4-y studies, and MUP amplitude and duration increased. In the last 2y period there was no further progression. Only 2y interval data is shown.

A

Table 1 .

 1 Neurophysiological studies:

This work was supported by "Fundação para a Ciência e Tecnologia" -PIC/IC/82765/2007.

Competing Interest: None declared (the authors have no conflict of interest).


Thomas Foltynie
email: t.foltynie@ion.ucl.ac.uk

Ludvic Zrinzo

Irene Martinez-Torres

Elina Tripoliti

Erika Petersen

Etienne Holl

Iciar Aviles-Olmos

Marjan Jahanshahi

Marwan Hariz

Patricia Limousin

Thomas Foltynie Box

MRI-guided STN DBS in Parkinson's disease without microelectrode recording: efficacy and safety

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a commonly employed therapeutic procedure for patients with Parkinson's disease uncontrolled by medical therapies. This series describes the outcomes of 79 consecutive patients that underwent bilateral STN DBS at the National Hospital for Neurology & Neurosurgery between November 2002 and November 2008 using an MRI-guided surgical technique without microelectrode recording (MER).

Patients underwent immediate postoperative stereotactic MR imaging. The mean (SD) error in electrode placement was 1.3 (0.6) mm. There were no haemorrhagic complications. At a median follow up period of 12 months, there was a mean improvement in the off-medication motor part of the Unified Parkinson´s Disease Rating Scale (UPDRS III) of 27.7 points (SD 13.8) equivalent to a mean improvement of 52% (p<0.0001). In addition there were significant improvements in dyskinesia duration, disability and pain, with a mean reduction in on-medication dyskinesia severity (sum of dyskinesia duration, disability and pain from UPDRS IV) from 3.15 (SD 2.33) pre-operatively, to 1.56 (SD 1.92) post operatively (p=0.0001). Quality of life improved by a mean of 5.5 points (median 7.9 points, SD 17.3) on the Parkinson's disease Questionnaire 39 (PDQ39) summary index. This series confirms that image-guided STN DBS without microelectrode recording can lead to substantial improvements in motor disability of well-selected PD patients with accompanying improvements in quality of life and most importantly, with very low morbidity.

"In clinical practice, brain imaging can now be divided in two parts: the diagnostic neuroradiology and the preoperative stereotactic localisation procedure. The latter is part of the therapeutic procedure. It is the surgeon's responsibility and should be closely integrated with the operation." Lars Leksell 1

Introduction

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an accepted surgical treatment for symptoms of Parkinson's disease (PD) inadequately controlled by medical therapies 2;3 . PD is a chronic neurodegenerative disease for which many medical treatments are available therefore interventions such as STN DBS, that are neither life-saving nor disease-modifying, but are aimed at improving quality of life, must meet the very highest standards of safety. Major risks associated with the surgical procedure include intra-cerebral haemorrhage and infection of the implanted material, while adverse effects of stimulation, are presumably related to stimulation of non-motor regions within or outside of the STN 4;5 .

Factors that may potentially improve safety and/or efficacy of the procedure for individual patients and for the group as a whole are of utmost importance.

The majority of centres currently use microelectrode recording (MER) from individual neurons to identify the characteristic neuronal firing patterns or 'physiological signature' of STN neurons during surgery.

Since the introduction and widespread adoption of STN DBS for PD, there has been a paucity of reports evaluating this procedure when it is performed without MER [START_REF] Patel | Magnetic resonance imaging-directed method for functional neurosurgery using implantable guide tubes[END_REF] . This study presents efficacy and safety outcomes of a large consecutive prospective series of PD patients (n=79) treated with STN DBS in a single centre, using a standardised surgical technique based on individual MRI-guided targeting without MER.

Material and Methods

Patients

Seventy-nine consecutive patients underwent STN DBS at the National Hospital for Neurology and Neurosurgery between November 2002 and November 2008. All patients met UK Brain Bank criteria for the diagnosis of PD and suffered from disabling motor complications of the illness despite optimal medical treatment. Prior to deciding on suitability for surgery, a brain MRI was obtained to exclude patients with advanced brain atrophy, white matter changes or other abnormality contra-indicating surgery. Baseline neuropsychological tests identified patients with significant cognitive impairment and L-dopa equivalent doses calculated for each patient [START_REF] Ch | Evolution of cognitive dysfunction in an incident Parkinson's disease cohort[END_REF] . An L-dopa challenge was performed to confirm drug responsiveness using the motor part of the Unified Parkinson´s Disease Rating Scale (UPDRS III).

A numerical measure of L-dopa response was derived as follows: (Off UPDRS III -On UPDRS III score) / Off UPDRS III x100. The off-medication state was practically defined as an overnight period free of medication. Speech was assessed using the Assessment of Intelligibility for Dysarthric Speech 8 . Patients were asked to complete the Parkinson's Disease Questionnaire 39 scale (PDQ39) [START_REF] Jenkinson | The Parkinson's Disease Questionnaire (PDQ-39): development and validation of a Parkinson's disease summary index score[END_REF] . The final decision regarding the appropriateness of STN DBS surgery for each patient was taken during a joint meeting of patient, immediate family, neurologist(s) and neurosurgeon(s).

Image acquisition and surgical procedure

Surgery was usually performed under local anaesthesia, in the off-medication condition, to allow clinical evaluation during electrode placement. If the patient was unable to tolerate prolonged periods offmedication, surgery was performed under general anaesthesia (n=12). The STN was visualised in each patient using specifically selected pre-operative stereotactic MRI sequences 10;11 following Leksell frame attachment ( © Elekta Instrument AB, Stockholm). Target selection for placement of the deepest contact was performed at the level of maximal rubral diameter (around 5mm below the AC PC plane) using commercially available planning software (FrameLink, Medtronic, Minneapolis). The STN was visualised as the hypointense signal lateral to the red nucleus. The entry point was defined at or behind the coronal suture to ensure a trajectory that would avoid sulci and the ventricular system. The exact entry and target point were subsequently modified to maintain a parenchymal trajectory whilst maximising the length of the trajectory within the visible STN hypointensity from the AC-PC to target level 12;13 . This was best achieved by reformatting images along the planned trajectory. As a result, the final target point was often located a couple of millimetres posterolateral to the target point described by Bejjani et al [START_REF] Bejjani | Bilateral subthalamic stimulation for Parkinson's disease by using threedimensional stereotactic magnetic resonance imaging and electrophysiological guidance[END_REF] .

(Figure 1). Since the sensorimotor STN is thought to occupy the supero-postero-lateral portion of the nucleus, this target point may bring the electrode trajectory closer to the desired functional target [START_REF] Yelnik | A three-dimensional, histological and deformable atlas of the human basal ganglia. I. Atlas construction based on immunohistochemical and MRI data[END_REF] .

During surgery, brain shift was avoided in several ways. Minimal CSF loss was achieved by placing the 14 mm burr hole and 3-4 mm small dural opening on a gyrus rather than a sulcus, flooding the burr hole with saline irrigation after dural opening, limiting the time from dural opening to final DBS electrode implantation and sealing the dural defect with fibrin glue as soon as the DBS electrode was in situ, and prior to test stimulation/ and or local field potential recording. Performing surgery in a similar position to that adopted during image acquisition, with only slight head-up tilt to encourage venous drainage, may further limit shift by minimizing postural movement of intracranial structures.

Dynamic impedance monitoring was performed while introducing a 1.5 or 2.2 mm diameter, blunt-tip radiofrequency (RF) electrode to the target (Leksell RF electrodes, Elekta, Stockholm). Great care was taken to avoid electrode deviation by contact with the burr hole or dural edges. A sharp tip probe would theoretically result in less brain deformation; however, the authors avoid this technique in view of the potential penetration, rather than displacement, of intraparenchymal vessels and resulting haemorrhage.

After withdrawal of the RF electrode, a quadripolar DBS electrode (Model 3389 DBS lead, Medtronic®, Minneapolis) was soft-passed down the same track. The depth of implantation of the deepest electrode was controlled by placing a depth stop a defined length along the electrode shaft. In those patients undergoing surgery under local anaesthesia, symptoms were assessed for the presence of a micro-lesion introduction effect. Monopolar stimulation through the contacts of the DBS electrode was then sequentially performed to assess for additional therapeutic effect and/or the presence of side effects (~10 minutes per side). The electrodes were secured in place with a skull fixation device (Medtronic burrhole cap or Stimloc device). Immediately following implantation of the DBS leads, all patients had a stereotactic MRI scan to confirm the electrode positions before implantation of the pulse generator. The perpendicular scalar (Euclidean) distance between the intended MRI target and the actual position of the implanted electrode was calculated on the immediate postoperative stereotactic MRI for each patient. The surgery was not considered to be finished until the post -operative imaging had confirmed acceptable placement of the electrodes. Prophylactic systemic antibiotics (Cefuroxime 1.5g) were administered intraoperatively and three more times during the following 24 hours. Frame fixation and imaging took approximately 45 minutes with an additional 30-45 minutes for stereotactic calculations. The typical duration of bilateral electrode implantation was 2 hours for surgery under general anaesthesia and 3 hours under local anaesthesia. All adverse events were recorded immediately post operatively and during subsequent follow up assessments.

Follow up

Selection of the optimal stimulation parameters was based on the acute clinical response to stimulation during the first post operative weeks, with further adjustment of parameters as required, at follow up visits typically at 1, 3, 6 and 12 months. To ensure optimal stimulation parameters, all patients underwent offand on-medication assessments at their 6-12 months post operative visit and then annually thereafter, using the UPDRS scale. Patients completed post-operative PDQ39 quality of life scales and neuropsychological assessments at their 6-12 month follow up visit, and follow up evaluation of speech was performed in all patients after 2004.

Data presentation and analysis

The presentation of clinical outcome data includes the median, the mean, and the range of outcomes.

Comparisons of baseline and post operative UPDRS & PDQ39 scores were performed using two sided sign tests to avoid assumptions regarding data distribution. The motor response to DBS was calculated in Imputation of data was performed among 8 patients who requested not to be assessed in post operative off-med, OFF-stim condition, as equal to baseline off-medication score. Composite subscores were derived from the sum of items 32-34 for total dyskinesia severity.

Results

Demographic data for the 79 consecutive patients are presented in Table 1.

Table 1

Demographic data of patients undergoing bilateral STN DBS.

Median, (Mean, SD, Range)

Gender distribution 49 male, 30 female Age at surgery 58.9 years, (57. L-dopa equivalent dose 1620 mg per day (1620, 641, 0-3260)

First degree relative diagnosed with PD 12/79 (15%)

Two patients had undergone unilateral pallidotomy prior to their initial consultations with us. In one of the 79 patients, the procedure was abandoned because of prolonged superficial bleeding from a cortical vein, and the consequent CSF leakage resulted in brain shift away from the skull. A decision was taken not to implant the permanent electrode after haemostasis was secured. Postoperative recovery was uneventful with no evidence of haemorrhage on postoperative imaging.

The remaining patients all underwent bilateral STN DBS, 77 during the same operating session. One patient had unplanned staged bilateral procedure because of a complex partial seizure that followed the insertion of the first electrode. Following surgery, two patients did not have follow up assessments because of 1) encephalopathy of unknown origin (presumed to be allergic) following electrode implantation requiring removal of all DBS hardware a few days later, and 2) death from previously undiagnosed asymptomatic carcinoma 9 months following surgery. The remaining 76 patients all underwent formal re-evaluation of PD severity after at least 6 months of post operative follow up. Where patients had multiple follow up assessments available, data was used from the assessment closest to the first anniversary of their surgery (median 12 months, mean 14 months (range 6-36 months).

All electrodes were implanted by means of a single brain penetration, except one electrode where two passes were needed. The median perpendicular targeting error between intended stereotactic coordinates and electrode trajectory, calculated from post operative stereotactic MR images, was 1.3 mm (mean 1.3 mm, SD 0.6, range 0 to 2.7 mm). An example of one of our patients' post operative stereotactic MRI scans is shown in Figure 1. The L-dopa equivalent dose at follow up was reduced by a mean of 633mg (39%). The mean stimulation parameters at follow up were: Left STN 3.0V, 60 microseconds, 139Hz;

Right STN 3.0V, 62 microseconds, 139Hz. Bipolar stimulation was used in 3/152 electrodes, monopolar stimulation for the remainder.

UPDRS

After a median follow up time of 12 months, the median improvement in UPDRS III comparing baseline off-medication scores to follow up off-medication, ON-stimulation scores was 56% (mean 52%, p<0.0001), see Table 2.

Table 2.

Comparisons of UPDRS III motor scores pre-operatively off-and on-medication, and post operatively off and on medication/stimulation. UPDRS-Unified Parkinson's disease rating scale. shown in Table 3. The median total dyskinesia severity score (sum of dyskinesia duration, disability & pain) was 3 pre-operatively (mean 3.15, SD 2.33), and 1 post operatively (mean 1.56 SD 1.92) equivalent to a reduction of 55% (p<0.0001).

Baseline

Table 3.

Comparison of UPDRS part IV sub-scores pre operatively and post operatively. 4. (These data were not routinely collected for the first 1 to 2 years of the period studied). For these individuals, DBS led to a median improvement of 7.9 points (mean 5.5 points, p =0.04) in the PDQ39 summary index, equivalent to an 18% improvement. There were improvements in the subscores; ADL, Stigma and bodily discomfort, and a trend towards worsening of communication.

Table 4.

Comparison of PDQ39 quality of life scores pre-operatively and post operatively. (SI-Summary Index = sum of change in each PDQ39 dimension/ 8, ADL-Activities of Daily Living).

Baseline

Speech Intelligibility

Pre and post operative assessments of speech intelligibility were performed for 49 consecutive patients.

The pre-operative mean "on medication" intelligibility score was 97.3 (SD 7.8). The follow up score "on medication & ON stimulation", had deteriorated to 84.4 (SD 27.2; p=0.0003).

Procedure & hardware related adverse events

Among this consecutive series of 79 patients, superficial bleeding from a cortical vein was encountered in one patient during surgery. Once haemostasis had been achieved, a substantial quantity of CSF had leaked from the dural opening, the brain had fallen away from the skull and a decision was taken not to implant the permanent electrode. The procedure was therefore abandoned. Postoperative recovery was uneventful with no new neurological deficit and no haemorrhage visible on post-procedure scan. A further patient had their electrodes removed within the first 2 weeks post operatively due to the development of a fluctuating encephalopathy with widespread white matter signal changes bilaterally following the course of the electrodes. The remaining "surgical" adverse events are documented in Table 6. One patient died from disseminated malignancy within 9 months of surgery and did not have follow up scoring performed.

Three patients underwent further surgery for their PD due to insufficient response from the first operation:

re-positioning of one of the two STN electrodes in one patient with improvement in clinical effect, and additional bilateral globus pallidus internus (GPi) electrode placement in two patients more than one year after their STN DBS procedure because of persisting severe painful dyskinesias/ dystonias. Neither of these patients has achieved good symptomatic control despite the additional surgery.

Table 6.

Adverse events arising as a result of surgery in this series.

Symptomatic haemorrhage Nil

Asymptomatic haemorrhage Nil

Pulmonary Embolus 1

Misplaced electrode 1

Pneumonia 1

Transient confusion/ delirium 7 Seizure 2

Suicide Nil

Scalp Erosion 1

Infection Nil

DBS Malfunction Nil

Lead fracture Nil

Lead migration Nil

Discussion

To the best of our knowledge, this is the largest published series of STN DBS performed in a single centre. Baseline patient demographics in the present study are similar to those operated in other series in terms of severity and duration of disease [START_REF] Deuschl | A randomized trial of deep-brain stimulation for Parkinson's disease[END_REF][START_REF] Weaver | Bilateral deep brain stimulation vs best medical therapy for patients with advanced Parkinson disease: a randomized controlled trial[END_REF][START_REF] Kleiner-Fisman | Subthalamic nucleus deep brain stimulation: summary and meta-analysis of outcomes[END_REF] . Median overall improvement from baseline in the UPDRS III off-medication motor score was 56% (mean 52%) and median improvement in PDQ39 Summary index was 7.9 points (mean 5.5 points). The low morbidity of the MRI-guided approach to surgery employed in this series demonstrates a high standard of safety while maintaining therapeutic efficacy.

There have been two large randomised controlled trials that evaluated the outcomes following DBS surgery in comparison to best medical treatment, one involving patients with STN DBS [START_REF] Deuschl | A randomized trial of deep-brain stimulation for Parkinson's disease[END_REF] and the other with patients having DBS in either the STN or GPi [START_REF] Weaver | Bilateral deep brain stimulation vs best medical therapy for patients with advanced Parkinson disease: a randomized controlled trial[END_REF] . Among 78 patients with STN DBS aged less than 75 years, there was a mean improvement in the off-medication UPDRS III motor score of 19.6 points (SD +/-15.1) , equivalent to a 41% improvement, comparing baseline to post operative scores at 6 months [START_REF] Deuschl | A randomized trial of deep-brain stimulation for Parkinson's disease[END_REF] .

In parallel with this there was a 9.5 point (SD +/-15.3) improvement in the PDQ39 summary index, equivalent to an overall mean improvement of 25% at 6 months, which is broadly similar to the overall mean improvement of 18% (median improvement 23.5%) in our patients after 1 year of follow up. This trial was conducted across 10 centres in Germany with STN targeting using either stereotactic MRI, ventriculography, microelectrode recording (MER) or a combination of these techniques according to local surgical protocols. Adverse events in this series included 1 death from an intra-cerebral haematoma, one suicide five months after surgery, and 2 infections at the stimulator site. The mean improvements in the clinical outcomes confirm the overall efficacy of the technique, although the wide standard deviation in mean outcome, as in the current series, suggests substantial inter-patient variability in response.

The second large randomised controlled trial of DBS [START_REF] Weaver | Bilateral deep brain stimulation vs best medical therapy for patients with advanced Parkinson disease: a randomized controlled trial[END_REF] included 121 patients who received either STN DBS (n=60) or GPi DBS (n=61). Of note is that this trial included at least 25% of the patients aged more than 70 years. All 13 participating sites were selected because they used MER to help in targeting.

Among the group of patients as a whole, there was a 12.3 point improvement (95%CI -14.3 to -10.3, SD not stated) in the UPDRS III off medication score at 6 months compared with baseline, equivalent to 29% improvement in scores. In addition there was a 7.7 point improvement in the PDQ39 summary index with improvements in 7/8 sub-domains. Among this group of patients, there was one patient death from intracerebral haemorrhage following the surgery, and 16 infections related to the electrodes or neurostimulator requiring removal of the hardware.

In a meta-analysis of open label studies including 21 patient populations [START_REF] Kleiner-Fisman | Subthalamic nucleus deep brain stimulation: summary and meta-analysis of outcomes[END_REF] , the mean improvement following STN DBS in the UPDRS motor subscale was 27.6 points (equivalent to 52% improvement from baseline), and the mean improvement in quality of life was 34.5%. These patients sustained intracranial haemorrhage in 3.9%, infection in 1.7%, and seizures in 1.5%. In a further meta-analysis including 31 STN studies and totalling 565 patients, an overall improvement of 54% in the UPDRS III score was identified [START_REF] Weaver | Deep brain stimulation in Parkinson disease: a metaanalysis of patient outcomes[END_REF] . The mean improvement in UPDRS III off-medication scores in the current series is thus in the average range when compared to that seen in either of the two large multi-centre trials 16;17 and the meta-analyses of published data 18;19 .

Image-guided surgery and surgical adverse events

The optimal target point within the visualised STN is as yet undefined. Our targeting strategy often resulted in the final target point being located a couple of millimetres posterolateral to the target point described by Bejjani et al [START_REF] Bejjani | Bilateral subthalamic stimulation for Parkinson's disease by using threedimensional stereotactic magnetic resonance imaging and electrophysiological guidance[END_REF] . (Figure 1). Since the sensorimotor STN is thought to occupy the most superior, posterior and lateral portion of the nucleus, a more posterolateral target point within the STN may bring the electrode trajectory closer to the functional target [START_REF] Yelnik | A three-dimensional, histological and deformable atlas of the human basal ganglia. I. Atlas construction based on immunohistochemical and MRI data[END_REF] .

Postoperative MRI confirmation of electrode location confirms the reliability of MRI-guided electrode placement without MER. There is a paucity of publications that report on outcome of STN DBS using image-guided techniques [START_REF] Patel | Magnetic resonance imaging-directed method for functional neurosurgery using implantable guide tubes[END_REF] . Our data demonstrates that MER may not be a pre-requisite to ensure wellplaced electrodes that deliver a satisfactory clinical outcome. Importantly, the adverse event profile seen in this series compares very favourably with that reported in neurosurgical series that used MER 17;18;20-28 , with no infection, haemorrhage or mortality associated with surgery, in the current series. The risk of symptomatic haemorrhage inevitably rises with increasing numbers of micro or macro electrode brain penetrations 20;21;23;29 . Furthermore, since MER significantly prolongs the procedure, we have been concerned that this potentially increases the risk of brain shift due to CSF leakage, which may paradoxically lower the precision of electrode placement as well as potentially increasing the risk of infection.

None of the patients in this series had visible haemorrhage on their post operative imaging. The single patient that suffered a pulmonary embolus was treated with a vena cava filter, made a complete recovery and has an excellent response to DBS. Of the two patients that had peri-operative seizures, one had a history of previous seizures the other did not. Neither patient is on long-term antiepileptic medication or has had seizures subsequently.

The number of surgical adverse events in our patients is very low. Nevertheless, the patient that required removal of electrodes warrants further discussion. Post-implant MR images revealed oedema surrounding the entire intra-cerebral course of both electrodes. This lady had no history of allergy, and did not show any evidence of nickel allergy when subsequently tested. The early post-operative course was unremarkable for the first few days but was followed by a fluctuating encephalopathy that did not respond to oral steroids. The electrodes were removed and were negative for Gram stain and fungal culture.

Subsequent management of PD symptoms has been with medical therapies alone. We are unaware of any previous publications of this type of reaction to DBS surgery.

Several patients in our series developed dysarthria limiting stimulation settings to those that provide more modest motor improvements. Outcome measures of improvement in motor disability using "optimal" stimulation parameters are thus also influenced by other confounders. In a previous analysis of speech outcomes following STN DBS we have reported that speech intelligibility deteriorates with stimulation among patients with electrodes situated in a more medial position [START_REF] Tripoliti | Effects of contact location and voltage amplitude on speech and movement in bilateral subthalamic nucleus deep brain stimulation[END_REF] . We therefore advocate imaging strategies that may further reduce the observed targeting error and improve reproducibility of electrode location with respect to the individual radiological anatomy.

This series confirms that MRI-guided STN DBS can lead to substantial mean improvements in motor disability of PD patients with improvement in quality of life. Reducing the risk associated with surgery through meticulous patient selection and optimal practice should serve as reassurance to patients with PD who may stand to benefit from this intervention but remain appropriately cautious about taking risks with their health.

Legend to

2 ways: 1 )

 21 Percentage improvement in UPDRS III motor score from baseline to follow up in the absence of medication = (Baseline off-med score) -(Post operative off-med ON-stim score) / (Baseline off-med score) x 100. 2) Percentage improvement in UPDRS III motor score by turning stimulation ON at follow up assessment in the absence of medication = (Post operative off-med, OFF-stim score) -(Post operative off-med, ONstim score) / (Post operative off-med, OFF-stim score) x 100.

  3, 7.7, 34.5-70.2) Duration of PD symptoms 11.5 years, (13.4, 7.0, 3.8-42.2)

  significant change in UPDRS III on-medication score. Comparison of baseline offmedication scores with follow up off-medication, OFF-stimulation scores shows a median decline of 1.5 UPDRS points (mean 3 points, range 28 points improvement to 35 points deterioration). Significant improvements were seen following STN DBS for UPDRS IV (complications of L-dopa treatment) as

3 PDQ39

 3 Completed PDQ39 quality of life data were available from both pre and post operative time periods in a subgroup of 49 consecutive individuals-Table

Figure 1 :

 1 Top panel: Preoperative stereotactic T2-weighted axial MRI (1.5T, 2mm thickness, no gap, TR 3500, TE 90.9) at the level of maximal rubral diameter (around 5mm below the AC PC plane). The subthalamic nucleus (STN) was visualized as the hypointense signal lateral to the red nucleus. Bottom left panel: The entry and target point were selected to maintain a parenchymal trajectory whilst maximising the length of the trajectory within the visible STN hypointensity from the AC-PC to target level. As a result, the final target point (open circle) was typically located 2.5 millimetres posterolateral to the target point originally described by Bejjani et al. (solid circle). Bottom right panel: Postoperative stereotactic T2-weighted axial MRI at equivalent level revealing electrode artefact at intended target.

Acknowledgements & Funding

This work was supported by the Parkinson's Appeal-registered charity 263064. The work was undertaken at UCLH/UCL who received a proportion of funding from the UK Department of Health's NIHR Biomedical Research Centres funding scheme.

The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive license (or non-exclusive for government employees) on a worldwide basis to the BMJ publishing group Ltd and its licensees to permit this article (if accepted) to be published in the Journal of Neurology, Neurosurgery & Psychiatry editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our license (http://jnnp.bmjjournlas.com/ifora/license.pdf).


T Tamme

M Reinik

T Püssa

M Roasto

K Meremäe

A Kiis

Dynamics of nitrate and nitrite content during storage of homemade and small-scale industrially produced raw vegetable juices and their dietary intake

Keywords: raw vegetable juice, nitrate, nitrite, storage, intake, ADI

The influence of storage conditions on nitrate and nitrite contents, pH and total viable bacterial count of raw vegetable juices was studied. Three different types of juices from an Estonian small-scale producer and five different types of homemade juices were analyzed.

Analyzes were performed immediately after opening the commercial juice packages and immediately after preparation of a homemade juice. Additionally, samples were taken after open storage of a juice at the refrigerator and ambient temperatures during 24 and 48 hours.

The biggest changes in nitrate and nitrite contents were found during the storage of carrot, beetroot and radish juices. During 48 hours of storage at ambient temperature the mean increase of nitrite content in homemade carrot, beetroot and radish juices were from 0.1 to 187, from 2.1 to 578 and from 0.5 to 259, respectively. In the case of commercial lightly pasteurized products, the biggest increase of nitrite content, from 3.2 to 11 mg l -1 was found in red beetroot juice. After 48 h of storage at refrigerator temperature the changes of nitrite and nitrate were smaller. In the case of consumption of 300 ml homemade carrot juice, with nitrate and nitrite content of 64 and 110 mg l -1 , respectively, stored for 24 h at ambient temperature, the average intake is 8 and 846% of ADI of nitrates and nitrites, respectively.

After consumption of 50 ml of the same carrot juice by children (1-2 years) the average intake of nitrates and nitrites is 7.0% and 733% of ADI, respectively.

Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online.

Introduction

Interest in the dietary intakes of nitrates and nitrites has arisen due to the concern about their possible adverse effect on health [START_REF] Belitz | Food Chemistry[END_REF][START_REF] Thomson | Intake and risk assessment of nitrate and nitrite from New Zealand foods and drinking water[END_REF][START_REF] Efsa -European | Nitrate in vegetables Scientific Opinion of the Panel on Contaminants in the Food Chain[END_REF].

Nitrate has a low level of acute toxicity but may be transformed into nitrite, which may lead to the formation of carcinogenic nitrosoamines [START_REF] Walker | Nitrates, nitrites and N-nitroso compounds: a Rewiew of the occurance in food and diet and the toxicological implications[END_REF][START_REF] Kolb | Potential Nitrosamine Formation and its Prevention During Biological Denitrification of Red Beet Juice[END_REF]) and the clinical symptoms of methaemoglobinemia [START_REF] Who | Evaluation of certain food additives and contaminants[END_REF][START_REF] Sanchez-Echaniz | Methemoglobinemia and Consumption of Vegetables in Infants[END_REF][START_REF] Santamaria | Nitrate in vegetables: toxicity, content, intake and EC regulation[END_REF]. Infant methemoglobinemia has been shown to be a result of consumption of high doses of nitrates by the infant foods and drinking water (Ezeagu, 1996, Coss et [START_REF] Thomson | Intake and risk assessment of nitrate and nitrite from New Zealand foods and drinking water[END_REF]). The acceptable daily intake (ADI) for nitrate is from 0 to 3.7 mg/kg body weight, and for nitrite from 0 to 0.06 mg/kg body weight, established by EU Scientific Committee for Food (EU Scientific Committee, 1995) and reconfirmed by JECFA in 2002.

Vegetable juices are an important source of essential nutrients for humans. During the last years the vegetable-based raw juices have become very popular. The popularity has arisen due to public interest in variable diets and alternative nutrition. Nitrate is naturally present in all vegetables and vegetable products that are substantial sources of nitrates in human intake. Some vegetables, such as carrot, pumpkin and cabbage, and particularly red beetroot and radish, can accumulate high levels of nitrates. Consumption of 150 ml of red beetroot juice containing 2000 mg l -1 nitrate thus would already result an intake greater than ADI value for a 70 kg adult [START_REF] Kolb | Potential Nitrosamine Formation and its Prevention During Biological Denitrification of Red Beet Juice[END_REF].

Nitrite content of most fresh, frozen or canned vegetables is generally low and usually in the range of 0 to 2 mg/kg [START_REF] Meah | Nitrate and nitrite in foods and the diet[END_REF], Pennington 1998[START_REF] Chung | Survey of nitrate and nitrite contents of vegetables grown in Korea[END_REF][START_REF] Belitz | Food Chemistry[END_REF][START_REF] Hsu | Nitrate and nitrite quantification from cured meat and vegetables an their estimated dietary intake in Australians[END_REF]. Reduction of nitrate to nitrite may take place in the presence of bacteria or enzyme nitrate reductase [START_REF] Phillips | Changes in the Nitrate and Nitrite Contents of Fresh and Processed Spinach during Storage[END_REF][START_REF] Chung | Changes in nitrate and nitrite content of four vegetables during storage at refrigerated and ambient temperatures[END_REF]) and after improper storage, especially of homemade vegetable products like purees and juices [START_REF] Heisler | Changes in Nitrate and Nitrite Content, and Search for Nitrosamines in Storage-Abused Spinach and Beets[END_REF][START_REF] Kolb | Potential Nitrosamine Formation and its Prevention During Biological Denitrification of Red Beet Juice[END_REF], Shanchez-Echaniz et al. 2001). It has been estimated that from 5 to 8% of the nitrate from the diet may be reduced to nitrite by the microflora in the oral cavity [START_REF] Mensinga | Health implications of exposure to environmental nitrogenous compounds[END_REF]) and up to 20% for individuals with a high rate of conversion [START_REF] Who | Evaluation of certain food additives and contaminants[END_REF].

If food contains high levels of nitrate, it is a potential risk if the conditions during storage or processing are conductive to conversion to nitrite [START_REF] Hill | Nitrates and nitrites from food and water in relation to human disease[END_REF][START_REF] Efsa -European | Nitrate in vegetables Scientific Opinion of the Panel on Contaminants in the Food Chain[END_REF]. Most critical are vegetables that have been damaged, poorly stored, or stored for extended periods, pickled and fermented vegetables as well as raw vegetables juices. In such circumstances, nitrite levels of up to 400 mg kg -1 have been found [START_REF] Who | Evaluation of certain food additives and contaminants[END_REF]. In fermented maize on storage in water at room temperature over period of 8 days, nitrate levels decreased 80% in average while the nitrite content increased about 200% in average of its initial level [START_REF] Ezeagu | Nitrate and nitrite contents in ogi and the changes occurring during storage[END_REF].

There is very limited up-to-date scientific information available on nitrate and nitrite levels and dynamics in the raw vegetable juices. According to the results of the earlier studies [START_REF] Phillips | Changes in the Nitrate and Nitrite Contents of Fresh and Processed Spinach during Storage[END_REF][START_REF] Heisler | Changes in Nitrate and Nitrite Content, and Search for Nitrosamines in Storage-Abused Spinach and Beets[END_REF]) the initial nitrate present in fresh vegetable juices was significantly reduced to nitrite under storage at ambient temperature. In 1994 Nabrzyski and Gajewska reported the decrease of nitrate from 261 to 46 mg kg -1 with consequent increase of During storage of homogenized leafy vegetables (spinach, crown daisy, etc.) at ambient temperature, nitrate levels in the vegetables dropped significantly (mean 87.4%) from the third day while nitrite levels increased dramatically in the range of 1857 to 3617 mg kg -1 . Refrigerated storage did not lead to significant changes in nitrate and nitrite levels in the homogenized leafy vegetables [START_REF] Chung | Changes in nitrate and nitrite content of four vegetables during storage at refrigerated and ambient temperatures[END_REF]. [START_REF] Phillips | Changes in the Nitrate and Nitrite Contents of Fresh and Processed Spinach during Storage[END_REF] reported that after storage of fresh homogenized spinach at ambient temperatures nitrate content was reduced to approximately 64% of its initial level.

Considerable public interest has lately been focused on the consumption of raw vegetable juices [START_REF] Yabsley | Miracle Juices[END_REF][START_REF] Kroom | Porgandi raviomadused[END_REF]. Different methods, like food supply data, published tables of the mean consumption of dietary items, duplicate portion analysis, dietary survey among the individuals, probabilistic and worse case screening models are used for estimation of the nitrate and nitrite intake from food and for calculations of the acceptable daily intakes [START_REF] Ellen | Dietary intakes of some essential and non-essential trace elements, nitrate, nitrite and N-nitrosamines, by Dutch adults: estimated via a 24-hour duplicate portion study[END_REF][START_REF] Penttilä | Nitrate, nitrite, and N-nitroso compounds in Finnish foods and the estimation of the dietary intakes[END_REF][START_REF] Murata | Survey of the daily intake of nitrate and nitrite in school children by the duplicate portion method[END_REF]. Depending on the method used, intake estimates can vary to a large extent [START_REF] Kroes | Assessment of intake from the diet[END_REF].

According to the Scientific Opinion of the Panel on Contaminants in the Food Chain of the European Food Safety Authority, adopted on 10 April 2008, there is a need for further research into the factors that influence nitrate and nitrite concentrations and alterations during production, storage and processing of the foodstuffs [START_REF] Efsa -European | Nitrate in vegetables Scientific Opinion of the Panel on Contaminants in the Food Chain[END_REF].

The aim of the present work was to study the changes of nitrate and nitrite levels in selected raw vegetables juices under refrigerated and ambient temperature storage conditions for a period of 2 days. The assessment of dietary nitrate and nitrite intake by consumption of raw vegetable juices in accordance with acceptable daily intakes (ADI) was the second objective of the present work.

Materials and methods

Juices

All juices were analysed in five replications.

Series 1 analyses were performed with lightly pasteurizated raw juices of carrot, cabbage and red beetroot, produced at a small-scale local enterprise and purchased at retail market. Light pasteurization is directed at vegetative microorganisms and is usually conducted at about 65 ºC during 10 seconds [START_REF] Sadler | Microbial, enzymatic, and chemical changes during storage of fresh and processed orange juice[END_REF]. The series 2 consisted of homemade raw juices prepared in the laboratory following the traditional raw juice preparation at homes without any pasteurization. Briefly, samples of five species of vegetables, i.e. carrot, cabbage, red beetroot, radish and pumpkin were collected from different outlets of Tartu, Estonia. The vegetables were washed with warm water, mechanically brushed and peeled. Raw juices were produced with commercially available squeezer (Stollar Juice Fountain).

All the raw juices were stored at two different temperatures, refrigerated (4-6 ºC) and ambient (20-22 ºC) for two days. The nitrate and nitrite contents, total viable counts, coliforms counts, yeast and moulds counts and pH were determined after opening the commercial juice packages and immediately after preparation of the homemade juices as well as after 24 and 48 hours of storage.

HPLC analysis of nitrate and nitrite ions

HPLC analyses were performed by a liquid chromatograph (Agilent Technologies Model 1100, Palo Alto, CA, USA) equipped with a fluorescence detector. Chromatographic separation of samples and standards was carried out at a reversed phase column Agilent 300SB-C 8 (2,1x150mm, 5µm; Agilent Technologies, USA) using gradient elution with 0.1% formic acid and acetonitrile eluent at 35 ºC with a speed of 0.2 ml/min. Fluorescence was monitored with excitation at 355 nm and emission at 420 nm.

Nitrate ions were converted to nitrite enzymatically by nitrate reductase and further to the highly fluorescent 2.3-naphthotriazole (NAT) with 2.3-diaminonaphthalene (DAN) as described in literature [START_REF] Misko | A Fluorometric Assay for the Measurment of Nitrite in Biological Samples[END_REF][START_REF] Li | Rapid determination of nitrite by reversed-phase highperformance liquid chromatography with fluorescence detection[END_REF]. Obtained solution was used directly for chromatographic analysis. Conversion rate of nitrate to nitrite was 98%, as detected with known amounts of nitrate and nitrite standards. All chemicals were purchased from Sigma, except sodium nitrate and potassium nitrite which were obtained from Riedel-de Haën. Limit of quantification (LOQ) of nitrate was 0.23 mg l -1 and of nitrite 0.10 mg l -1 .

Bacteriological analyses

All juices were examined microbiologically for total viable counts, coliform bacteria, yeasts and moulds using the four plate method. Decimal dilution series were prepared in accordance with EVS-EN ISO 6887-1:2001. Plate Count Agar LAB 115 was used for isolation of total viable counts at 30 °C for 72 h (EVS-EN ISO 4833:2006). Coliforms were grown in the Violet Red Bile Agar LAB 31 (V.R.B.A.) at 37 °C for 24 h (NMKL 44 5th edition 2001).

Yeast extract-dextrose-chloramphenicol-agar medium LAB 119-B5 was used for isolation of yeast and moulds at 25 °C for 5 days (EVS-EN ISO 7954:1999). Microbiological counts were expressed as a number of colony forming units (cfu) per milliliter (ml).

Determination of pH-values of vegetable juices

The pH of the juices was determined by pH meter Consort C 833, according to the instructions of European Standard EVS-EN 1132:2000.

Methods of intake estimation

The consumption amounts of raw vegetable juices were calculated in accordance with dietary recommendations for average intake of 65 kg body weight of adult by [START_REF] Kroom | Porgandi raviomadused[END_REF]. Intake of nitrates and nitrites by infants and young children was estimated in accordance with the nutrition recommendations of Estonian paediatricians [START_REF] Kroom | Porgandi raviomadused[END_REF].

Statistical analysis

All individual laboratory results of nitrate and nitrite content were recorded and the statistical analyses were performed using MS Excel 2003 software (Microsoft Corporation; Redmond, WA, USA) T-test was used to reveal the statistical significance of differences in measured variables at different time points. The results with values p<0.05 were considered statistically significant. Correlation analyses were used to study relationships between the pH and total viable count of aerobic bacteria (log cfu/ml) as well as between nitrate/nitrite content and total viable count of bacteria (log cfu/ml).

Results and discussion

The range and mean values of initial nitrate and nitrite contents in the studied vegetable-based juices are shown in Table 1. The highest mean initial values of nitrate were obtained for the homemade juices of beetroot and radish, 2625 and 4615 mg l -1 , respectively. Significantly lower values were determined for the carrot juices (p<0.05). The mean nitrite content for all juices was below 3.2 mg l -1 . 2. Nitrate content decreased in all three juices during storage at both temperatures. The biggest decrease in nitrate content were found during 48 hours of storage for carrot, cabbage and beetroot juices at the ambient temperature, 47% (87 to 46 mg l -1 ), 39% (250 to 152 mg l -1 ), 57% (1708 to 739 mg l -1 ), respectively. After 48 hours of storage at refrigerated temperatures smaller decreases in nitrate contents was found, 11-30% in comparison with the initial contents. The biggest increase of nitrites, from 3.2 to 11.1 mg l -1 and from 3.2 to 6.8 mg l -1 was found for raw beetroot juice during storage both at ambient and refrigerated temperatures, respectively (Table 2). The pH dynamics of the juice samples is shown in Table 2. In the carrot juice, stored during 48 h at ambient temperatures, the highest pH decrease, from 6.3 to 4.1, was measured. The pH changes were lower in juices, stored at refrigerator temperatures.

[Insert Table 2 about here] Series 2. The changes in nitrate and nitrite mean contents in the homemade carrot, cabbage, radish and pumpkin raw juices are shown in Figure 1. During the storage the nitrate contents in juices were decreasing and conversely, the nitrite contents were increasing. Changes in nitrate and nitrite contents depend on the juice type and storage conditions. Storage of the homemade raw juices for 24 h at ambient temperatures is clearly unacceptable, because the nitrite contents in carrot, beetroot and radish juices increases significantly (p<0.05) (Figure 1 A, B, D). Also, there was statistically significant increase of nitrite content (Figure 1 E) in cabbage juices (p<0.05). At the same time the nitrate contents decreased during 24 hour storage in the range of 26-66% of the initial contents. The biggest decrease in nitrate content was found after 24 h storage at ambient temperatures for carrot and beetroot juices, from 163 to 64 mg l -1 and from 2625 to 692 mg l -1 , respectively (Table 4, Figure 1 A,B). Nitrate contents decreased during 48 h of storage at ambient temperatures for carrot, beetroot and radish raw juices, from 163 to 6.7 mg l -1 , 2625 to 523 mg l -1 and 4615 to 1772 mg l -1 , respectively (Figure 1 A,B,D). At the same time, there was a considerable increase in nitrite content from 0.1 to 187 mg l -1 , from 2.1 to 578 mg l -1 and from 0.5 to 259 mg l -1 was registered, respectively (Figure 1 A, B, D). For example, during 48 hours of storage of the cabbage juice at the ambient temperature the nitrate contents decreased from 116 to 44 mg l -1 , at the same time the nitrite contents stayed below 2.3 mg l -1 (Figure 1 C). In general, during 48 h of storage at refrigerated temperatures the nitrate contents decreased. The biggest decreases, from 163 to 108 mg l -1 and from 2625 to 1486 were found for carrot and beetroot raw juices, respectively (Figure 1 A,B). At the same time, during the 48 h storage at refrigerated temperatures, nitrite contents increased mostly for carrot juice, 47 mg l -1 and less for beetroot and radish juice, 4.9 and 11.2 mg l -1 , respectively (Figure 1).

In an earlier study, [START_REF] Heisler | Changes in Nitrate and Nitrite Content, and Search for Nitrosamines in Storage-Abused Spinach and Beets[END_REF] reported a considerable decrease in nitrate and increase of nitrite content during 24 hours at ambient temperature for the beetroot and spinach raw juices. The nitrite content of beetroot and spinach juices increased up to 540 and 900-1000 mg kg -1 , respectively. The nitrate content decreased from 3800 to 2800 and from 1500 to 350 mg kg -1 , respectively [START_REF] Heisler | Changes in Nitrate and Nitrite Content, and Search for Nitrosamines in Storage-Abused Spinach and Beets[END_REF].

The pH dynamics of the raw juice samples is illustrated in Figure 1. The pH of the juices did not change considerably during the storage at refrigerated temperatures. Differently, at ambient temperatures, the highest decrease of pH value from initial 6.4 to 4.2 was observed in radish juice. The pH of the raw juice samples was measured at 0, 24 and 48 h of storage. Generally, during the storage studies, the pH decreased and nitrite contents and total viable counts increased (Figure 1). Since both nitrite concentration and total viable microbial counts increased during storage it may be concluded that microbial activity was the main factor in nitrate reduction process. Better correlations between the nitrite content and total viable counts were found in vegetable juices after storage at ambient temperatures (r = 0.9 to 0.58) It can be explained by microbiological reduction of nitrite from nitrate ion. The negative correlations between nitrate content and total viable counts were found from homemade carrot juice and homemade radish juice (r = -0.84 to -0.81).

It is recommended to use only fresh-prepared homemade carrot juices for babies. Raw material used for the preparation of juices should have as low initial nitrate content as possible.

[Insert Figure 1

Microbiological count and pH value.

All the products were analyzed for total viable bacterial, coliform bacteria, yeasts and moulds counts. In the series 1, three different vegetable-based small-scale industry produced raw juices and in series 2, five different homemade vegetable-based raw juices were microbiologically analyzed. During the storage, all the species of the microorganisms showed the increase in counts (Table 3 andFigure 1), while pH showed decrease tendency (Table 2 and Figure 1). Between the pH and total viable counts (log cfu/ml) clear negative correlations were determined (r=-0.95 to -0.75, p<0.05). The decrease of pH is caused by the microorganisms' vital activity products, as indicated by the increase of microorganism multiplicity. As reported by [START_REF] Akin | A model for pH determination during alcoholic fermentation of a grape must by Saccharomyces cerevisiae[END_REF], pH decrease was assumed to correlate with the nitrogen consumption of the studied microorganism strains. The nitrogen concentration did not influence the pH itself but during the fermentation, the consumption of nitrogen by yeasts produces H + ions [START_REF] Castrillo | Proton production and consumption pathways in yeast metabolism. A chemostat culture analysis[END_REF]. In Table 3 it is shown that compared to initial microorganism counts, after 48 hours of storage at ambient temperature the average percentage in counts of microorganisms in homemade raw juices were higher than for commercially produced juices, 173 and 161%, respectively. During the storage experiment of the industrial lightly pasteurized raw juices at ambient temperatures for 48 h the bacterial total counts increased from 5.03-5.33 log cfu/ml to 7.81-8.85 log cfu/ml. During the storage of the homemade juices at ambient temperature for 48 h the total bacterial counts increased from 5.14-5.39 log cfu/ml to 8.84-9.19 log cfu/ml, with the average increase 3.8 log cfu/ml.

During storage of raw juices at ambient temperatures, the total counts of coliform bacteria and moulds and yeast increased significantly (p<0.05). Higher increase of microorganism counts was detected in the homemade juices.

[Insert Table 3 about here]

Intake calculation of nitrate and nitrite

Adults. The intake of nitrate and nitrite has been calculated in accordance with the recommendations given by Estonian and other dietitians [START_REF] Heinerman | Heinerman`s encyclopedia of juice cures[END_REF], Yabsley and Gross, 2001[START_REF] Kroom | Porgandi raviomadused[END_REF]. The recommended consumption data of the homemade vegetable based raw juices and calculated ADI percentages are shown in Table 4. In calculations of nitrate and nitrite daily intakes, 65 kg was used as the average body weight of Estonian adults. The average consumption amounts of vegetable raw juices in accordance with dietitians' recommendations were 100 to 300 ml dependent on the juice variety. The daily doses of nitrate and nitrite depend on initial contents of NO 3 -and NO 2 -in vegetable based raw juices (Table 4). Additionally, it depends on consumption amounts and storage conditions of the vegetable-based raw juices. The daily doses are exceeded by adults even by consumption of freshly made beetroot and radish juices as well as by using the same juices after 24 h of storage at refrigerated temperatures, 164%, 193% and 109%, 172% of ADI, respectively (Table 4). Daily doses of nitrites when juices are stored at same conditions are generally low and in range of 0.8% to 16% of ADI, exceptional was a carrot juice with 30% of ADI. Nitrite daily doses increased significantly (p<0.05) when the juices were stored at ambient temperatures for 24 h, at the same time the nitrate doses decreased. Nitrite doses in the case of consumption of 300 ml raw carrot juice, 150 ml raw beetroot juice and 100 ml of raw radish juice exceeded ADI values 846%, 346% and 167%, respectively. Beetroot and radish juices could be mixed with other vegetable based raw juices as recommended by dietitians [START_REF] Heinerman | Heinerman`s encyclopedia of juice cures[END_REF][START_REF] Kroom | Porgandi raviomadused[END_REF]. Industrially produced raw juices are lightly pasteurized and therefore the nitrate and nitrite changes (Table 2) are lower compared to the homemade juices (Table 4). Consequently, the nitrate and nitrite daily doses are also considerably lower and will not exceed the ADI values.

[Insert Table 4 about here]

Infants and young children. According to our data obtained from questionnaires, filled in by parents, the most popular raw vegetable juices given to young children were carrot based raw juices. Generally, carrot juices contain nitrates in low concentrations. In our study the carrot raw juices produced by Estonian small-scale industry and in homemade juices contained nitrates 87 and 163 mg l -1 , respectively (Table 2 and4).

Recommended consumption of raw carrot juices and related nitrates and nitrites daily doses are shown in Table 5. In accordance with our data, it is obvious that only freshly made carrot raw juices can be used for infants because the ADI values of nitrate and nitrite were 12.9% and 0.5%, respectively. Consumption of the fresh and 24 h at refrigerated temperatures stored homemade carrot raw juices increased nitrite daily intakes to 19.7% of ADI. During 24 h of storage at ambient temperatures of the homemade carrot raw juices the nitrite contents were higher compare to storage in refrigerator which resulted with the mean values of 110 mg l -1 (Table 4). Consumption of a juice containing 110 mg l -1 of nitrite by 10-12 months-old infants 30 ml as the maximum amount recommended by dieticians and 50 ml for 1-2 years old young children could be harmful for health because the mean nitrite intake is 540% and 733% of ADI, respectively (Table 5). According to the calculations by [START_REF] Phillips | Changes in the Nitrate and Nitrite Contents of Fresh and Processed Spinach during Storage[END_REF], one dose of raw vegetable juice in amount of 30 ml could result in cyanosis for a 1-year-old child.

Lightly pasteurized carrot raw juice (Table 2) made in small-scale industry could be given for young child even after 24 h of storage at refrigerated temperatures because as shown in Table 2 the nitrate and nitrite contents had only minor changes, 87 mg l -1 to 80 mg l -1 and 0.1 mg l -1 to 0.2 mg l -1 , respectively. In the present case the mean daily nitrate dose for infants in result of consumption of carrot raw juices will stay below 10% of ADI and nitrite dose will not exceed 2% of ADI.

[Insert Table 5 about here]

Conclusions

The initial contents and changes in the dynamics of nitrates and nitrites were determined after 24 and 48 hours of storage at ambient and refrigerated temperatures of homemade (carrot, beetroot, cabbage, radish, pumpkin) and commercially produced raw vegetable based juices (carrot, beetroot, cabbage). A significant decrease (p<0.05) in nitrate and a significant increase (p<0.05) in nitrite contents in homemade carrot, beetroot and radish raw juices after 48 hours of storage at ambient temperatures was found. Nitrite contents of carrot, beetroot and radish raw juices were high already after 24 h of storage at ambient temperatures.

Therefore, the consumption of the homemade vegetable based raw juices is recommended only shortly after preparation. The industrially produced lightly pasteurized raw juices can be openly stored at refrigerated temperatures for a maximum of 24 hours. In conclusion, very high nitrite intakes from vegetable based raw juices can occur when juices are stored for a long time at an improper e.g. at ambient temperatures. Acceptable daily intakes can be exceeded 5-8 fold for young children and 2-8 fold for the adults.

F

  14 to 83.0 mg kg -1 for juices prepared from blanched carrot and stored at ambient temperatures.

  and pH value.Storage experiments were performed at ambient (20-22 °C) and refrigerator (4-6 ºC) temperatures during 24 and 48 hours.Series 1. The changes in nitrate and nitrite mean contents in a small-scale company produced slightly pasteurized juices during open storage are shown in Table

Figure 1 .

 1 Figure 1. The changes of nitrate and nitrite content during storage in raw homemade vegetables juices -carrot juice A, beetroot juice B, cabbage juice C, radish juice D, pumpkin juice E

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Table 1 .

 1 The changes of nitrate and nitrite content during storage in raw homemade vegetables juicescarrot juice A, beetroot juice B, cabbage juice C, radish juice D, pumpkin juice E Nitrate and nitrite initial contents of raw vegetable juices, 5 replications

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Table 2 .

 2 The changes of mean nitrate and nitrite content during storage of commercial raw vegetable juice (5 replications) at ambient (20-22°C) and refrigerated (4-6°C) temperature

	Type of	Storage	Nitrate mg l -1	Nitrite mg l -1		pH
	juice	period (hours)	20-22°C	4-6°C	20-22°C	4-6°C	20-22°C	4-6°C
	Carrot	0	87	87	0.1	0.1	6.3	6.3
	juice	24	63	80	0.4	0.2	5.3	6.3
		48	46	62	2.7	1.5	4.1	6.2
	Cabbage	0	250	250	0.6	0.6	6.0	6.0
	juice	24	192	236	1.3	0.6	5.0	5.7
		48	152	188	1.5	1.2	4.2	5.5
	Red	0	1708	1708	3.2	3.2	5.9	5.9
	beetroot	24	1158	1606	7.1	4.7	5.0	5.8
	juice	48	739	1517	11.1	6.8	4.2	5.7

Table 4 .

 4 The mean content of nitrate and nitrite and changes during storage in raw homemade vegetable juices and daily intake by Estonian adults Vegetable juice

				Food Additives and Contaminants
		Nitrate	Nitrite	Recommended	Expected	% of	Expected	% of
		content	content	mean intake of	nitrate intake	ADI	nitrite intake	ADI
		mg l -1	mg l -1	juice per day, ml*	mg day -1		mg day -1
	Raw juices before storage
	Carrot	163	0.1	300	49.0	20.4 0.03	0.8
	Cabbage 116	0.23	200	23.0	9.7	0.05	1.2
	Red	2625	2.1	150	394.0	164.0 0.32	8
	beetroot
	Radish	4615	0.5	100	462.0	193.0 0.05	1.3
	Pumpkin 255	1.0	200	51.0	21.3 0.2	5.1
	Raw juices after storage at refrigeration temperature (4-6 ºC) within 24 h
	Carrot	152	3.9	300	45.6	19.0 1.17	30
	Cabbage 100	0.4	200	20.0	8.3	0.08	2.1
	Red	1745	2.5	150	262.0	109.0 0.38	9.6
	beetroot
	Radish	4126	1.5	100	413.0	172.0 0.15	3.8
	Pumpkin 230	3.1	200	46.0	19.2 0.62	16
	Raw juices storage at ambient temperature (20-22 ºC) within 24 h
	Carrot	64	110.0 300	19.0	8.0	33.0	846
	Cabbage 65	1.2	200	13.0	5.4	0.24	6.2
	Red	692	90.0	150	103.8	43.3 13.4	346
	beetroot
	Radish	2633	65.1	100	263.0	109.7 6.5	167
	Pumpkin 167	14.3	200	33.4	13.9 2.86	73

* calculated by the basis of the recommended by dietitians.

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Table 5 .

 5 Estimated mean nitrate and nitrite intake by infants and young children after consumption of raw home-made carrot juice 12 months infants, mean body weight 10.2 kg -recommended consumption of raw carrot juice 30 ml per day b 1 -2 year young children, mean body weight 12.5 kg -recommended consumption of raw carrot juice 50 ml per day

	Storage condition Fresh raw juice Stored 24 h at 4-6°C Stored 24 h at 20-22°C a 10 -Page 21 of 20	NO 3 content -mg l -1 163 152 64	NO 2 content -mg l -1 0.1 3.9 110	Mean NO 3 intake mg per day 4.9 a 8.2 b 4.6 a 7.6 b 1.9 a 3.2 b	-	% of ADI 12.9 a 17.8 b 12.0 a 16.5 b 5.0 a 7.0 b	Mean NO 2 intake mg per -day 0.003 a 0.005 b 0.12 a 0.20 b 3.3 a 5.5 b	% of ADI 0.5 a 0.7 b 19.7 a 26.6 b 540 a 733 b

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Food Additives and Contaminants

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants