Preclinical quality, safety, and efficacy of a human embryonic stem cell-derived product for the treatment of Parkinson's disease, STEM-PD.

Cell replacement therapies for Parkinson's disease (PD) based on transplantation of pluripotent stem cell-derived dopaminergic neurons are now entering clinical trials. Here, we present quality, safety, and efficacy data supporting the first-in-human STEM-PD phase I/IIa clinical trial along with the trial design. The STEM-PD product was manufactured under GMP and quality tested in vitro and in vivo to meet regulatory requirements. Importantly, no adverse effects were observed upon testing of the product in a 39-week rat GLP safety study for toxicity, tumorigenicity, and biodistribution, and a non-GLP efficacy study confirmed that the transplanted cells mediated full functional recovery in a pre-clinical rat model of PD. We further observed highly comparable efficacy results between two different GMP batches, verifying that the product can be serially manufactured. A fully in vivo-tested batch of STEM-PD is now being used in a clinical trial of 8 patients with moderate PD, initiated in 2022.

A Perspective of International Collaboration Through Web-Based Telecommunication-Inspired by COVID-19 Crisis.

The tsunami effect of the COVID-19 pandemic is affecting many aspects of scientific activities. Multidisciplinary experimental studies with international collaborators are hindered by the closing of the national borders, logistic issues due to lockdown, quarantine restrictions, and social distancing requirements. The full impact of this crisis on science is not clear yet, but the above-mentioned issues have most certainly restrained academic research activities. Sharing innovative solutions between researchers is in high demand in this situation. The aim of this paper is to share our successful practice of using web-based communication and remote control software for real-time long-distance control of brain stimulation. This solution may guide and encourage researchers to cope with restrictions and has the potential to help expanding international collaborations by lowering travel time and costs.

Non-enzymatic transformations of dietary 2-hydroxyalkenyl and aromatic glucosinolates in the stomach of monogastrics.

Monogastric animals exhibit different biological responses to structurally diverse glucosinolates and their transformation products, depending on the dietary levels. The transformations of 2-hydroxyalkenyl and aromatic glucosinolates were examined in vitro under gastric conditions, ex vivo in ligated porcine stomachs and in vivo in a rat model. Intact glucosinolates were almost completely transformed in vitro within 1 h at pH 3 (73-88%) and at pH 5 (97-100%) upon addition of Fe2+ ranging from two-fold molar excess. Glucosinolate transformations reached 78-99% when incubated ex vivo in ligated porcine stomachs. Rat in vivo feeding trials showed major reductions (81-84%) in the intact glucosinolate contents upon passage through the gastrointestinal (GI) tract. Non-enzymatic transformations of glucosinolates occur in the stomach, where pH and the level of Fe2+ are primary determinants. This is the first study to show a complex formation between iron-progoitrin and iron-sinalbin, facilitating the transformation into nitriles and thionamides.

Age as a prognostic factor in relation to surgical evacuation of spontaneous supratentorial intracerebral haemorrhage.

AIM AND CLINICAL RATIONALE FOR THE STUDY: Spontaneous intracerebral haemorrhage (sICH) is an acute life-threatening injury and constitutes 10-15% of first-ever stroke cases. The Surgical Trials in Intracerebral Haematoma studies (STICH and STICH II) represent the two foremost studies in the field, however, with arguable shortcomings. To find more accurate criteria, we aimed to correlate the preoperative neurological and neuroimaging findings with the clinical outcome of operated patients. MATERIALS AND METHODS: In this retrospective study, sICH patients were recruited from the Central Denmark Region from 2010 to 2016. We evaluated the patients' medical records regarding preoperative Glasgow Coma Scale (GCS) 6 months and one year after surgery, focal neurological defects, thrombolytic treatment, pupil status, and haemorrhage localization visualized by neuroimaging. The patients' clinical outcome was assessed using the Glasgow Outcome Scale (GOS). RESULTS: Based on logistic multiple linear analysis, age, basal ganglia haemorrhage and mass effect had significant effect on the mortality rate. Besides, age, basal ganglia haemorrhage, intra ventricular haemorrhage and pupil difference had significant correlation with good outcome (GOS>3). CONCLUSIONS AND CLINICAL IMPLICATIONS: Neurosurgical treatment of the sICH patients is indicated only if age and potentially improved morbidity is carefully evaluated considering the STICH and this study; otherwise, we will just increase the health care burden with a number of extremely care-dependent patients.

High pressure effects on myrosinase activity and glucosinolate preservation in seedlings of Brussels sprouts.

Combinations of pressure, temperature and time (100-600 MPa, 30-60 °C, 3-10 min) influence enzyme activity of the myrosinase-glucosinolate system. Seedlings of Brussels sprouts were used as a model, which constitutes a well-defined and homogenous sample matrix with simple cell structures. A response surface methodology approach was used to determine the combined effect of pressure level, temperature and time on glucosinolate concentration and myrosinase activity in Brussels sprouts seedlings. The effects on residual myrosinase activity and intact glucosinolate concentration differed according to combinations of pressure, time and temperature. The results showed that maximum inactivation of myrosinase and preservation of glucosinolate (85% of the untreated level) was obtained after HP treatment at 600 MPa, 60 °C, 10 min. The highest preservation of myrosinase activity compared to untreated seedlings was after HP at 100 MPa, 30 °C, 3 min and 10 min with low degree of cell permeabilization.

Brain Tissue Reaction to Deep Brain Stimulation-A Longitudinal Study of DBS in the Goettingen Minipig.

OBJECTIVES: The use of Deep Brain Stimulation (DBS) in treatment of various brain disorders is constantly growing; however, the number of studies of the reaction of the brain tissue toward implanted leads is still limited. Therefore, the aim of our study was to analyze the impact of DBS leads on brain tissue in a large animal model using minipigs. METHODS: Twelve female animals, one control and eleven with bilaterally implanted DBS electrodes were used in our experiment. 3, 6, and 12 months after implantation the animals were sacrificed, perfused and the brains were removed. Tissue blocks containing the lead tracks were dissected, frozen, sectioned into 40 µm sections and stained using Nissl and Eosin, anti-GFAPab or Isolectin. The tissue reaction was analyzed at five levels, following from the distal lead tip, to compare tissue response in stimulated and nonstimulated areas: four segments along each level of electrodes, and the fifth level lying outside the electrode area (control area). The sections were described both qualitatively and quantitatively. Quantitative assessment of the reaction to the implanted electrode was based on the measurement of the area covered by the staining and the thickness of the glial scar. RESULTS AND CONCLUSIONS: Tissue reaction was, on average, limited to distance of 500 µm from the lead track. The tissue response after 12 months was weaker than after 6 months confirming that it stabilizes over a time. There was no histological evidence that the stimulated part of the electrode triggered different tissue response than its nonstimulated part.

The Retrograde Connections and Anatomical Segregation of the Göttingen Minipig Nucleus Accumbens.

Nucleus accumbens (NAcc) has been implicated in several psychiatric disorders such as treatment resistant depression (TRD), and obsessive-compulsive disorder (OCD), and has been an ongoing experimental target for deep brain stimulation (DBS) in both rats and humans. In order to translate basic scientific results from rodents to the human setting a large animal model is needed to thoroughly study the effect of such therapeutic interventions. The aim of the study was, accordingly, to describe the basic anatomy of the Göttingen minipig NAcc and its retrograde connections. Tracing was carried out by MRI-guided stereotactic unilateral fluorogold injections in the NAcc of Göttingen minipigs. After 2 weeks the brains were sectioned and subsequently stained with Nissl-, autometallographic (AMG) development of myelin, and DARPP-32 and calbindin immunohistochemistry. The minipig NAcc was divided in a central core and an outer medial, ventral and lateral shell. We confirmed the NAcc to be a large and well-segregated structure toward its medial, ventral and lateral borders. The fluorogold tracing revealed inputs to NAcc from the medial parts of the prefrontal cortex, BA 25 (subgenual cortex), insula bilaterally, amygdala, the CA1-region of hippocampus, entorhinal cortex, subiculum, paraventricular and anterior parts of thalamus, dorsomedial parts of hypothalamus, substantia nigra, ventral tegmental area (VTA), the retrorubral field and the dorsal and median raphe nuclei. In conclusion the Göttingen minipig NAcc is a large ventral striatal structure that can be divided into a core and shell with prominent afferent connections from several subrhinal and infra-/prelimbic brain areas.

Testing a reality orientation program in patients with traumatic brain injury in a neurointensive care unit.

PRIMARY OBJECTIVE: The aim of this study was to examine the effect of a systematic reality orientation program (RO) introduced in a neurointensive care unit on duration of posttraumatic amnesia (PTA) and outcomes of patients with traumatic brain injury (TBI). RESEARCH DESIGN: This study used a quasiexperimental, prospective design. METHODS AND PROCEDURES: Twenty-four patients (intervention) with a significant TBI classified as moderate-to-severe injuries as measured by scores of less than 12 on the Glasgow Coma Scale underwent an RO program compared with a similar group of 38 patients (control) who received a conventional rehabilitation program. The Rancho Los Amigos Score was used to assess the cognitive level 24 hours after the end of sedation, and the Galveston Orientation and Amnesia Test was used daily to assess orientation and duration of PTA. The Glasgow Outcome Scale Extended was then used as an indicator of clinical outcome after 12 months. MAIN OUTCOMES AND RESULTS: The preliminary results indicated that patients who received the RO had a higher mean of the Glasgow Outcome Scale Extended (SD = 1.53) than those receiving the usual care (SD = 1.35) despite that the groups differed significantly (p = .01) in PTA duration. CONCLUSION: Patients with TBI may benefit from early assessment and systematic RO nursing intervention. The RO may facilitate patients with PTA to regain orientation and interact with their surroundings in the neurointensive care unit to optimize the recovery. However, further studies with focus on timing, intensity, and duration are needed to evaluate the influence of an early RO approach on PTA and outcomes in patients experiencing TBI.

Glyphosate spray drift in Coffea arabica - sensitivity of coffee plants and possible use of shikimic acid as a biomarker for glyphosate exposure.

Glyphosate is widely used in coffee plantations to control weeds. Lacking selectivity, glyphosate spray drift is suspected to cause adverse effects in coffee plants. Symptoms caused by glyphosate can be similar to those produced by other stress factors. However, shikimic acid accumulation should be a useful biomarker for glyphosate exposure as shown for other crops. The aim of this study was to assess the sensitivity of coffee plants towards glyphosate on different biological response variables and to evaluate the use of shikimic acid as biomarker. Dose-response experiments yielded ED50 values (50% effect dose) in the range of 38-550 ga.e.ha(-1) depending on the quantitative or qualitative variable monitored. The frequency of plants showing symptoms was the most sensitive variable. The best sampling time for shikimic acid accumulation was 1-2 weeks after glyphosate application, depending on experimental conditions. The highest shikimic acid accumulation was observed in young leaves. Shikimic acid is a suitable biomarker for a glyphosate exposure in coffee, using only young leaves for the analysis. Young coffee plants are susceptible to glyphosate damage. If symptoms are absent the risk of severe crop damage or yield loss is low.

Capillary transit time heterogeneity and flow-metabolism coupling after traumatic brain injury.

Most patients who die after traumatic brain injury (TBI) show evidence of ischemic brain damage. Nevertheless, it has proven difficult to demonstrate cerebral ischemia in TBI patients. After TBI, both global and localized changes in cerebral blood flow (CBF) are observed, depending on the extent of diffuse brain swelling and the size and location of contusions and hematoma. These changes vary considerably over time, with most TBI patients showing reduced CBF during the first 12 hours after injury, then hyperperfusion, and in some patients vasospasms before CBF eventually normalizes. This apparent neurovascular uncoupling has been ascribed to mitochondrial dysfunction, hindered oxygen diffusion into tissue, or microthrombosis. Capillary compression by astrocytic endfeet swelling is observed in biopsies acquired from TBI patients. In animal models, elevated intracranial pressure compresses capillaries, causing redistribution of capillary flows into patterns argued to cause functional shunting of oxygenated blood through the capillary bed. We used a biophysical model of oxygen transport in tissue to examine how capillary flow disturbances may contribute to the profound changes in CBF after TBI. The analysis suggests that elevated capillary transit time heterogeneity can cause critical reductions in oxygen availability in the absence of 'classic' ischemia. We discuss diagnostic and therapeutic consequences of these predictions.

Potential of cultivar and crop management to affect phytochemical content in winter-grown sprouting broccoli (Brassica oleracea L. var. italica).

BACKGROUND: Variety and crop management strategies affect the content of bioactive compounds (phenolics, flavonoids and glucosinolates) in green broccoli (calabrese) types, which are cultivated during summer and autumn in temperate European climates. Sprouting broccoli types are morphologically distinct and are grown over the winter season and harvested until early spring. Thus they show considerable potential for development as an import substitution crop for growers and consumers during the 'hungry gap' of early spring. The present study investigated the effect of variety and management practices on phytochemical content in a range of sprouting broccoli varieties. RESULTS: Yields were significantly higher in white sprouting broccoli varieties. Levels of phenolics and flavonoids were in the range 81.64-297.65 and 16.95-104.80 mg 100 g⁻¹ fresh weight, respectively, depending on year and cultivar, and were highest in variety 'TZ 5052' in both years. In-row spacing did not affect flavonoid content. Phenolic and flavonoid content generally increased with increasing floret maturity and levels were high in edible portions of the crop. Crop wastes (leaf and flower) contained 145.9-239.3 and 21.5-116.6 mg 100 g⁻¹ fresh weight total phenolics and flavonoids, respectively, depending on cultivar, tissue and year. Climatic factors had a significant effect on phenolic and flavonoid content. Levels of total and some individual glucosinolates were higher in sprouting broccoli than in the green broccoli variety 'Ironman'. CONCLUSION: Levels of total phenolics, flavonoids and glucosinolates are higher in sprouting than green broccoli types. Sprouting broccoli represents an excellent source of dietary bioactive compounds.

Reduced areas of spontaneous neuropathic pain after spinal cord stimulation treatment.

OBJECTIVES: Spinal cord stimulation (SCS) is known to be an effective treatment for a range of neuropathic pain (NeP) conditions, although further clinical evidence is required. Clinical observations suggest that 1 aspect of the treatment effect is a reduction in the area with spontaneous NeP. The aim of this study was to quantify the areas of spontaneous NeP in SCS patients and to correlate these with changes in pain intensity and quality of life. METHODS: Twenty-six SCS patients with NeP rated their pain intensity on a numerical rating scale (0 to 10) and completed the SF-36 health survey. In addition, their areas of spontaneous pain were photographically documented before and during SCS treatment, and the areas were transferred to phantom drawings and digitally quantified. RESULTS: Areas of spontaneous NeP were reduced by 62% (interquartile range, -15 to 78). Pain intensity was reduced from 7.5 ± 1.1 before to 4.9 ± 1.7 during treatment (mean ± SD) and most domains of the SF-36 health survey also improved with SCS treatment. Using linear regression, no correlation was found between relative reduction in areas of spontaneous NeP and relative reduction in pain intensity as well as in absolute improvement in quality of life assessed by SF-36. A correlation was found between improvement in pain intensity and in quality of life. DISCUSSION: The results indicate that the main impact of SCS on the patients' quality of life is not the reduction of the painful areas, but rather the reduced pain intensity in the remaining area.

Recurrence of chronic subdural haematomata with and without post-operative drainage.

Chronic subdural haematoma is a common disease causing morbidity and mortality. Recurrence after surgical treatment is common, varying from 5% to 30% of cases. Several methods for reducing recurrence have been advocated. The aim of this study was to investigate the effect of post-operative subdural drainage. Three hundred forty-four patients were included in a retrospective study. Treatment was performed by burr hole irrigation. Groups were separated into those with post-operative drainage compared to those without. Recurrence occurred in 14% in the drained group, significantly less than 26% in the undrained group (p=0.011). There were no differences in the complication rates. Post-operative drainage reduces recurrence of chronic subdural haematoma without increasing the complication rate. These results support those reported in several other studies. We recommend the use of post-operative subdural drainage.

A critical review of the literature on early rehabilitation of patients with post-traumatic amnesia in acute care.

AIMS AND OBJECTIVES: To identify the effect of early rehabilitation of patients with post-traumatic amnesia in patients with traumatic brain injury, to review and critically assess evidence related to the timing of intervention and to identify the effect on outcome of a specific neurological rehabilitation in acute care. BACKGROUND: Up to 70% of patients with traumatic brain injury will experience post-traumatic amnesia. Although duration of post-traumatic amnesia is correlated negatively with outcome for patients with traumatic brain injury, there is limited evidence relating to what influence timing and effect may have on enhancing early rehabilitation patient outcomes. DESIGN: A critical literature review. METHODS: Searches for systematic reviews were undertaken in Medline, CINAHL, Cochrane, PSYC INFO and Neurotraume databases. The efficacy of intervention and timing was classified based on a hierarchy of study designs for questions about health care interventions based on soundness of design. RESULTS: Six reviews and 11 original studies were included and comprised the review. Many studies used weak designs and small sample size, thus limiting their ability to control confusing variables and outcomes. Few studies included papers with the information about timing and effect of early post-traumatic amnesia intervention. Only one study showed an effect of a reality orientation programme in acute care. Although there was no significant detail reported on the possibility of reducing the post-traumatic amnesia period, the study showed clinical relevance. CONCLUSION: This review highlights the limited evidence of the effect of early rehabilitation of patients with post-traumatic amnesia. Future research should be conducted to identify the effectiveness of early intervention. RELEVANCE TO CLINICAL PRACTICE: Although nurses are treating patients with post-traumatic amnesia without systematic assessment, the limited evidence available does little to direct nurses as to the best approach to start early rehabilitation of post-traumatic amnesia to promote good outcomes.

Fe2+-catalyzed formation of nitriles and thionamides from intact glucosinolates.

The ratio of isothiocyanates to nitriles formed upon the hydrolysis of glucosinolates is a key factor determining the physiological effect of glucosinolate-containing plants and materials. A micellar electrokinetic capillary chromatography (MECC) method was used to study the nonenzymatic Fe2+-catalyzed transformation of glucosinolates. At room temperature, pH 5, and in the presence of only 2 molar excess of Fe2+ all glucosinolate was degraded in 24 h. At all molar excess Fe2+ tested, nitriles were the major compounds formed. Thionamides were also formed from glucosinolates that contained a side chain hydroxylated at C-2; in this case, trace amounts of oxazolidine-2-thione were also detected. The presence of Fe3+ had no effect. The nonenzymatic Fe2+-catalyzed transformation of glucosinolates involves the binding of Fe2+ to the glucosinolate to form a complex.

The biosynthetic pathway for aurofusarin in Fusarium graminearum reveals a close link between the naphthoquinones and naphthopyrones.

Fungal polyketide biosynthesis typically involves multiple enzymatic steps and the encoding genes are often found in gene clusters. A gene cluster containing PKS12, the polyketide synthase gene responsible for the synthesis of the pigment aurofusarin, was analysed by gene replacement using Agrobacterium tumefaciens-mediated transformation to determine the biosynthesis pathway of aurofusarin. Replacement of aurR1 with hygB shows that it encodes a positively acting transcription factor that is required for the full expression of PKS12, aurJ, aurF, gip1 and FG02329.1, which belong to the gene cluster. AurR1 and PKS12 deletion mutants are unable to produce aurofusarin and rubrofusarin. Bio- and chemoinformatics combined with chemical analysis of replacement mutants (DeltaaurJ, DeltaaurF, Deltagip1, DeltaaurO and DeltaPKS12) indicate a five-step enzyme catalysed pathway for the biosynthesis of aurofusarin, with rubrofusarin as an intermediate. This links the biosynthesis of naphthopyrones and naphthoquinones together. Replacement of the putative transcription factor aurR2 results in an increased level of rubrofusarin relative to aurofusarin. Gip1, a putative laccase, is proposed to be responsible for the dimerization of two oxidized rubrofusarin molecules resulting in the formation of aurofusarin.

Micellar electrokinetic capillary chromatography--synchronous monitoring of substrate and products in the myrosinase catalysed hydrolysis of glucosinolates.

A micellar electrokinetic capillary chromatography (MECC) method has been developed for monitoring the myrosinase catalysed hydrolysis of 2-hydroxy substituted glucosinolates and the simultaneous formation of the corresponding degradation products (oxazolidine-2-thiones (OZTs) and nitriles). Glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate) was chosen as the model glucosinolate owing to the difficulties in determining hydrolysis rates of this type of substrates in traditional UV-assays. The method was afterwards validated with glucobarbarin ((2S)-2-hydroxy-2-phenylethylglucosinolate) and progoitrin ((2R)-2-hydroxybut-3-enylglucosinolate). Aromatic glucosinolates without a 2-hydroxy group in their side chains, such as glucotropaeolin (benzylglucosinolate) and gluconasturtiin (phenethylglucosinolate) were also tested. Formation of the glucosinolate hydrolysis products was monitored simultaneously at 206 nm and 230 nm. This allowed estimation of the extinction coefficient of the OZT derived from glucosibarin, which was found to be 18,000 M(-1) cm(-1) and 12,000 M(-1) cm(-1) at 206 nm and 230 nm, respectively. The developed method has limit of detection of 0.04 mM and 0.06 mM and limit of quantification of 0.2 mM and 0.3 mM for the glucosibarin derived OZT and nitrile, respectively. Linearity of the glucosinolate concentration was examined at six concentration levels from 2.5 mM to 100 mM and at 206 nm a straight line (R(2)=0.9996) was obtained. The number of theoretical plates (N) at the optimal system conditions was 245,000 for the intact glucosibarin, 264,000 for the OZT and 252,000 for the nitrile.

Therapeutic strategies for neurodegenerative disorders: emerging clues from Parkinson's disease.

Our knowledge of Parkinson's disease pathophysiology has greatly expanded during the last century, resulting in successful new medical and neurosurgical approaches toward this common neurodegenerative disorder. These approaches might also be useable in the treatment of psychiatric disorders, which often are linked to atrophic and degenerative processes in the brain; however, the successful application of these techniques in psychiatry requires thorough elucidation of disease pathophysiology to identify proper intervention sites. Likewise, awareness of the differences between the parkinsonian and psychiatric patient populations in terms of age, disease course, and life expectancy, as well as ethical considerations might in the end determine the appropriateness of these therapeutic strategies in psychiatry.

The anatomy of the porcine subthalamic nucleus evaluated with immunohistochemistry and design-based stereology.

This study provides a light-microscopic description of the organization, morphology and number of neurons in the subthalamic nucleus (STN) of the Göttingen minipig. It is based on histological material stained with Nissl, Golgi and autometallographic techniques, and employs design-based stereological estimation of the total neuron number. The organization of several neurotransmitters in the STN has been evaluated in histological preparations stained for acetylcholinesterase (AChE) and immunostained for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and glutamate. In all of the stained preparations the STN appeared as a distinct lens-shaped structure located in the caudal diencephalon, medial to the internal capsule and ventrolateral to the zona incerta. Rostrally, the STN approached the globus pallidus pars interna, whereas caudally the ventromedial part of the STN was adjacent to the rostral part of the substantia nigra pars compacta (SNc), where some of the neurons of the two nuclei merged. The neurons in the STN had medium-sized (25-40 microm) ovoid or fusiform cell bodies, from which three to six large dendrites emanated in a direction predominantly parallel to the long axis of the STN. Immunohistochemistry revealed that most of the subthalamic neurons were glutamatergic and differed significantly in appearance from the large stellate TH-positive cells of the adjacent SNc. Numerous TH-positive bouton-rich fibers traversed the STN. The GAD-staining revealed a large number of terminals within the boundaries of the STN. The STN was highly AChE-positive, reflecting a prominent innervation by ChAT-positive terminals. The total number of subthalamic neurons in one hemisphere was estimated to be approximately 56,000. We conclude that the neuroarchitecture of the porcine STN is similar to primates, including humans, and appears well-suited for further studies examining the role of the STN in movement disorders.

High-performance capillary electrophoresis with indirect UV detection for determination of alpha-galactosides in Leguminosae and Brassicaceae.

A rapid, easy, and reproducible capillary electrophoresis method for determination of raffinose family oligosaccharides (alpha-galactosides) was developed. Sucrose, raffinose, stachyose, verbascose, and ajugose were determined with indirect UV detection at moderate alkaline pH 9.2, using pyridine-2,6-dicarboxylic acid as background electrolyte in a sodium tetraborate buffer with added cetyltrimethylammonium bromide. The separation efficiency measured by the number of theoretical plates (N) ranged from 1.4 x 10(5) to 2.3 x 10(5). The precision of the method, measured by the relative standard deviation (RSD), was less than 0.53% for the migration times and better than 3.4% for normalized areas (NA), considering all sugars except verbascose (RSD(NA) = 11.8%). Detection limits were about 110 microg/mL, corresponding to 150-320 microM. Relative response factors (RRF) were calculated on the basis of linearity studies and used for quantification of alpha-galactosides in a lupine sample (Lupinus angustifolius).