Early and intensive Motor Training for people with spinal cord injuries (the SCI-MT Trial): description of the intervention.

STUDY DESIGN: Descriptive. OBJECTIVES: The primary objective is to describe the intervention that will be provided in a large multi-centre randomised controlled trial titled: Early and Intensive Motor Training for people with Spinal Cord Injuries (the SCI-MT Trial). The secondary objective is to describe the strategies that will be used to operationalise and standardise the Motor Training provided to participants while keeping the intervention person-centred. METHODS: The paper focuses on the rationale and principles of Motor Training for people with spinal cord injuries (SCI). The description of the intervention is based on the Template for Intervention Description and Replication (TIDieR) checklist. Specifically, it addresses the following 6 criteria of the TIDieR checklist: why the effectiveness of Motor Training is being examined; what, how, where and when the Motor Training will be administered; and how much Motor Training will be provided. RESULTS: A detailed intervention manual has been developed to help standardise the delivery of the intervention. CONCLUSIONS: This paper describes the details of a complex intervention administered as part of a large randomised controlled trial. It will facilitate the subsequent interpretation of the trial results and enable the intervention to be reproduced in clinical practice and future trials.

Do people with multiple sclerosis perceive upper limb improvements from robotic-mediated therapy? A mixed methods study.

BACKGROUND: Robot-mediated training is increasingly considered as a rehabilitation intervention targeting upper limb disability. However, experiences of such an intervention have been rarely explored in the multiple sclerosis population. This mixed methods study sought to explore the impact of an eight week upper limb robotic intervention on experiences of people with multiple sclerosis. METHODS: Eleven participants (Median EDSS- score: 6.5) with moderate to severe upper limb impairment, performed eight week robot- mediated training of the most affected arm. The training involved a virtual learning platform called I-TRAVLE with duration of 30 min per training session, twice to three times per week. Two subjective questionnaires with items from the Intrinsic Motivation Inventory (IMI) and Credibility and Expectancy Questionnaire (CEQ) were collected bi-weekly during the intervention. Approximately one month after completing the training, three focus groups were conducted. Main themes were identified using thematic analysis. RESULTS: Results from the IMI and CEQ indicated high patient satisfaction and expectation that the intervention will be beneficial for them. Two main themes were identified: (1) Effect of the intervention on impairment and activity in that participants felt that there was a positive impact of the training on strength, endurance and during activities of daily living and that it met their expectations; (2) Experiences based on system usage from the system in that participants expressed feelings of motivation and self-improvement. The participants also perceived the training as enjoyable and concluded that the use of games instigated a competitive spirit between the participants. CONCLUSIONS: Robot-mediated training could have a motivating effect and induce a general positive experience while reducing disabilities of people with multiple sclerosis.

Outcome of revised metal-on-metal hip arthroplasties: a Dutch arthroplasty register study.

BACKGROUND: Preliminary results of metal-on-metal (MoM) hip arthroplasty were satisfactory, but since 2004 data showed high failure rates. National joint replacement registries are multi-centre databases comprised of thousands of subjects and implants which allow for identifying variables predictive of implant failure. The aim of the current study was to estimate re-revision rates after revision of a primary MoM hip arthroplasty in the Dutch Arthroplasty Register (LROI) and to assess potential predictor variables of re-revision of these MoM hip arthroplasties. METHODS: Eligible procedures were those with a revision for any reason except infection, after an initial primary surgery with a hip resurfacing (HRA) or large-head MoM (LH-MoM) total hip arthroplasty (THA). The probability of re-revision for both types of MoM hip arthroplasty over time was estimated using the cumulative incidence function taking mortality as a competing risk into account. A proportional sub-distribution hazards regression model was used to assess potential predictor variables of re-revision of these MoM hip arthroplasties. RESULTS: A total of 3476 records of revised implants were included, of which 873 (25.2%) were MoM implants. Over the course of follow-up, 101 (11.5%) MoM implants were re-revised. During follow-up 36 (4.3%) patients who received a MoM-implant at primary arthroplasty and a revision afterwards had died. The regression model showed that for primary MoM implants a MoM articulation after revision (HR 2.48; 95% CI 1.53-4.03, p < 0.001), femoral-only revisions (HR 3.20; 95% CI 2.06-4.99, p < 0.001) and periprosthetic fractures (HR 1.98; 95% CI 1.03-3.82, p = 0.042) as reason for the first revision were statistically significant risk factors for re-revision. CONCLUSION: Both types of large-head MoM hip arthroplasties have shown high revision and re-revision rates; risk factors were identified. The outcome of this study can be helpful in managing expectations of patients and orthopaedic surgeons.

Risk factors for total ankle arthroplasty failure: A Dutch Arthroplasty Register study.

BACKGROUND: Studies concerning total ankle arthroplasty could be influenced by several forms of bias. Independent national arthroplasty registries represent objective data on survival and patient reported outcomes. The aim of this study was to determine survival and identify risk factors for early failure in a nationwide series of total ankle arthroplasties from the Dutch Arthroplasty Register (LROI). PATIENTS AND METHODS: Data of 810 patients, who received 836 total ankle arthroplasties between 2014 and 2020 were obtained from the Dutch Arthroplasty Register (LROI) with a median follow-up of 38 months (range 1-84 months). Survival was expressed in Kaplan-Meier analysis and associated hazard ratios for implant failure were determined. Implant failure was defined as the need for revision surgery for any reason or (pan)arthrodesis. RESULTS: During follow-up, we recorded 39 failures (4.7%) resulting in a implant survival of 95.3% with a median follow-up of 38 months (range 1-84 months). Medial malleolus osteotomy (HR = 2.27), previous surgery (HR = 1.83), previous osteotomy (HR = 2.82) and previous ligament reconstruction (HR = 2.83) all showed potentially clinically meaningful associations with a higher incidence of implant failure, yet only previous OCD treatment (HR = 6.21), BMI (HR = 1.09) and age (HR = 0.71) were statistically significant. INTERPRETATION: Excellent short-term survival (95.3%) with a median follow-up of 38 months was reported for TAA patients from the Dutch Arthroplasty Register. Patients with a lower age, a higher BMI or who had a prior surgical OCD treatment before TAA surgery appear to have a higher risk for revision after short-term clinical follow-up. Thorough patient selection with emphasis on risk factors associated with early implant failure might be essential to improve TAA survivorship.

A tool to enhance occupational therapy reasoning from ICF perspective: The Hasselt Occupational Performance Profile (H-OPP).

BACKGROUND: In order to enhance occupational therapy reasoning in clinical practice, different elements such as client-centred approach, evidence-based care and interdisciplinary work should be taken into account, but is a challenge. AIM: To describe the development of the digital Hasselt Occupational Performance Profile (H-OPP©) that enhances occupational therapy reasoning from ICF perspective. METHOD: A participative qualitative design was used to create the H-OPP© in an iterative way in which occupational therapy lectures, ICF experts, students and occupational therapists in the field were involved. RESULTS: After linking occupational therapy terminology to the ICF, different stages of the H-OPP were identified and elaborated with main features: generating an occupational performance profile based on inventarization of problems and possibilities, formulating an occupational performance diagnosis and enabling to create an intervention plan. In all stages, both the perspectives of the client and the occupational therapist were taken into account. To increase practical use, the tool was further elaborated and digitalized. CONCLUSION: The H-OPP© is a digital coach that guides and facilitates professional reasoning in (novice) occupational therapists. It augments involvement of the client system. Furthermore, it enhances interdisciplinary communication and evidence-based care.

Robot-assisted task-oriented upper extremity skill training in cervical spinal cord injury: a feasibility study.

STUDY DESIGN: Prospective multiple case study. OBJECTIVES: To test (1) the feasibility of haptic robot technology (Haptic Master (HM)) use to improve arm-hand function (AHF) and arm-hand skill performance (AHSP) in persons with a cervical spinal cord injury (C-SCI), (2) inventory participants' motivation and expectation to work with the robot technology used and (3) to descriptively report the results in individual cases. SETTING: Rehabilitation Centre. METHODS: Five C-SCI patients were trained for 6 weeks, 3 days per week, 60 min per day. Therapists filled out the Usefulness, Satisfaction and Ease-of-use questionnaire (USE). The Intrinsic Motivational Inventory (IMI) and credibility and expectancy questionnaire (CEQ) were filled out by participants. Performance at activity level was gauged using the Van Lieshout test for AHF in Tetraplegia and the Spinal Cord Independence Measure. Function level was gauged using muscle strength testing and the International Classification for Surgery of the Hand in Tetraplegia. RESULTS: As to the feasibility of the application of haptic robot technology, the mean USE score was 65%. Mean IMI and CEQ results were 67% and 60%, respectively. Participants were motivated to train with the HM. All participants rated credibility higher than expectations regarding the improvement. In the current patients, little progress was demonstrated at the International Classification of Functioning, Disability and Health function and the activity level. CONCLUSION: It is feasible to train C-SCI persons with the HM. Therapists report that working with the HM is easy to learn and easy to perform. Usability of the HM may be improved. Further research is needed to assess in which group of C-SCI and at which stage of rehabilitation HM training may be most beneficial.

Reference values for the transformed Van Lieshout hand function test for tetraplegia.

STUDY DESIGN: Longitudinal cohort study. OBJECTIVES: Previously, the Van Lieshout hand function test for tetraplegia (short form)(VLT-SF) has been transformed into an interval scale (r_VLT-SF) using the Rasch analysis, thereby fulfilling the requirements of an objective measurement. The present study aims (1) to transform r_VLT-SFlogit scores (r_VLT-SFlogit) into 0-100 scores (r_VLT-SF100) in order to facilitate communication amongst its users and (2) to describe r_VLT-SF100 reference values in order to refine the prediction of upper extremity skilled performance in persons with tetraplegia. SETTING: Eight Dutch rehabilitation centres. METHODS: The VLT-SF data of tetraplegic patients from eight Dutch rehabilitation centres, gathered at the start of the active rehabilitation (t1), 3 months after t1 (t2) and at the time of discharge (t3), have been used. The r_VLT-SF100 total score for t1 and t3 were computed, anchored on the data for t2. Reference values (medians and interquartiles) for different subgroups were calculated at different moments during the rehabilitation. RESULTS: Data at t1 (n=64), t2 (n=73) and t3 (n=69) were used. The r_VLT-SFlogit scores (-4.78-6.32) were transformed into r_VLT-SF100 (0-100) at t2. Reference values were established for persons with motor complete or incomplete lesions with either a high (C1-C6) or a low (C7-T1) lesion level at t1, t2 and t3. Significant differences (P<0.025) in r_VLT-SF100 were found for most subgroups and different time intervals. CONCLUSIONS: The r_VLT-SFlogit scores were converted into r_VLT-SF100 scores, and reference values of the r_VLT-SF100 have been established for different subgroups of tetraplegic patients at different stages during rehabilitation. The r_VLT-SF100 has been proven to be able to detect improvement over time.

Improvement of the Van Lieshout hand function test for tetraplegia using a Rasch analysis.

STUDY DESIGN: Cross-sectional study. OBJECTIVE: The Van Lieshout hand function test for tetraplegia (VLT) measures the quality of arm-hand functioning in persons with tetraplegia. It is valid, reliable and responsive. However, it does not satisfy all the criteria for interval level measurement. The present study aims to apply the Rasch model to the VLT short form (VLT-SF) to upgrade its scale type towards interval level, and to verify if the requirements of an objective measure are satisfied in the revised version. SETTING: Eight Dutch Rehabilitation centres. METHODS: The VLT-SF data of 73 tetraplegic patients were Rasch-analysed (RUMM2030 software, RUMM Laboratory Pty Ltd, Perth, Australia) to verify the order of response categories, unidimensionality and reliability of the VLT-SF, and to assess its applicability regardless of (motor) lesion completeness. RESULTS: Seven of the ten VLT-SF items showed disordered response categories. The six original response categories were therefore recoded into three or four categories. After recoding, all items satisfied the model requirement of unidimensionality. The items were relatively well-targeted on the subjects' arm-hand skilled performance measures, leading to a good person separation index (R=0.91). The difficulty hierarchy of the VLT-SF items was invariant across patient subgroups of (motor) lesion completeness. CONCLUSIONS: Provided that response categories are recoded, VLT-SF Rasch analysis showed that the requirements of an objective measure were satisfied. This allows to compare the measurements of different patients quantitatively, and to follow their results over time.

Arm hand skilled performance in persons with a cervical spinal cord injury--long-term follow-up.

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: To assess development of arm hand skilled performance (AHSP) during and after in-patient rehabilitation in persons with cervical spinal cord injury (CSCI) and to determine factors that influence the outcome. SETTING: Eight rehabilitation centres in the Netherlands with specialised spinal cord injury departments. METHODS: AHSP was assessed using the Van Lieshout test (VLT) in persons admitted with recent CSCI. Assessment was carried out at the beginning (t1), after 3 months (t2), at the end (t3) of in-patient rehabilitation, and 1 and 5 years thereafter (t4, t5). Multilevel regression analysis was performed to determine development of AHSP and associations between AHSP and age, gender, motor completeness, lesion level (high or low CSCI), motor scores of upper extremity (MSUE), and pain in the tested arm. RESULTS: Fifty-five participants were included with mean age 38 years (range 18­64). There were 73% male, 80% had high CSCI (C3­C6) and 69% had motor complete lesion. Scores of VLT improved significantly during in-patient rehabilitation (mean: t1=25; t3=33) (P=0.005), scores remained unchanged at 1 year (t4=32) and 5 years (t5=32) (P=0.903) after in-patient rehabilitation. Motor completeness, MSUE and pain were significantly related to the VLT score (P<0.001, P<0.001, P=0.015, respectively). Age, gender and lesion level had no significant relationship. CONCLUSION: AHSP improved during in-patient rehabilitation. It was then stable during the next 5 years after discharge. Persons with an incomplete lesion, high MSUE and no pain in the tested arm perform best on the VLT.

ToCUEST: a task-oriented client-centered training module to improve upper extremity skilled performance in cervical spinal cord-injured persons.

OBJECTIVES: Given (a) the importance of upper extremity skill performance in persons with cervical spinal cord injury (C-SCI), (b) the limited number of studies on arm hand functioning in C-SCI and (c) the discrepancy between the importance of client centeredness and the lack of a theoretical framework and practical guidelines on how to apply client centeredness into practice, the aim of the present study is to (1) develop a training concept in which rehabilitation of the upper extremity focuses on patient's needs and wishes and (2) to put this concept into practice for tetraplegic persons. METHODS: The training concept contains two main components. The client-centered component includes both extracting individual goals and defining and objectifying these goals. The task-oriented component incorporates a task analysis and the composition of an individual training program. RESULTS: A task-oriented client-centered training aimed at improving upper extremity skilled performance in tetraplegic persons is described step by step. The Canadian Occupational Performance Measure is used to identify patient's specific needs. The Goal Attainment Scale is applied to define and objectify individual goals. A task analysis is essential to map limiting and facilitating factors of the person, the task and the environment and to identify which aspects should be trained. An individual task-oriented training program is composed, based on principles of motor learning and training physiology and the use of assistive devices. Each step is explained and illustrated using a case example.

Evaluation of a task-oriented client-centered upper extremity skilled performance training module in persons with tetraplegia.

STUDY DESIGN: Clinical intervention study. OBJECTIVE: Arm hand skilled performance (AHSP) has a major role in the rehabilitation of persons with cervical spinal cord injury (C-SCI). A task-oriented client-centered upper extremity skilled performance-training (ToCUEST) module was developed. The present study aims to evaluate effects of ToCUEST on specific and general AHSP in C-SCI, during and after rehabilitation. SETTING: Adelante Rehabilitation Centre (The Netherlands). METHODS: C-SCI persons, either during or after rehabilitation, completed the ToCUEST training; a control group of patients during rehabilitation received standard rehabilitation. The training module was focused on patient's individual needs, combining principles of motor learning and training physiology. Three self-selected goals were trained 3 days per week, in three sessions of 30 min per day for 8 weeks. Measures were taken at 3 months after the start of the active rehabilitation (T0), before training (T1), after training (T2), at follow-up (3 months post-training; T3) and at discharge. RESULTS: For both ToCUEST groups during (n=11) and after (n=12) rehabilitation, an improvement (P<0.001) on specific AHSP (using the Goal Attainment Scale and the Canadian Occupational Performance Measure (satisfaction and performance)) was found between T1-T2 and T1-T3. Also an improvement (P<0.02) in general AHSP (using the Van Lieshout Test, the QIF and the motor FIM) was found in the total ToCUEST group between T1-T2 and T1-T3. No significant difference in improvement of general AHSP was found between the ToCUEST rehab and control group. CONCLUSION: The ToCUEST module leads to improvement in AHSP, not only persons with C-SCI during rehabilitation, but also after finishing rehabilitation. These effects remain at follow-up.

Measuring change in arm hand skilled performance in persons with a cervical spinal cord injury: responsiveness of the Van Lieshout Test.

OBJECTIVES: To asses whether the Van Lieshout Test (VLT) is responsive to measure changes in arm hand skilled performance (AHSP) over time during active rehabilitation in persons with cervical spinal cord injury (C-SCI) and in different subgroups of persons with C-SCI according to lesion completeness and to lesion level. STUDY DESIGN: Longitudinal cohort study. SETTING: Spinal Cord Injury Units in eight rehabilitation centres across the Netherlands. METHODS: In the present study, data from a national SCI cohort study are used. Data from the VLT, the Grasp Release Test (GRT), the Functional Independence Measure (FIM) and the Quadriplegia Index of Function (QIF) were recorded at three moments during active rehabilitation: at the start of active rehabilitation (t1), 3 months after t1 (t2) and at discharge (t3). Data have been analysed using three responsiveness measures, that is, the standardized response mean, the effect size and comparison of test scores measured at different times. RESULTS: The VLT is responsive for the intervals t1-3, t1-2 and t2-3. The VLT can be used to measure changes in AHSP in persons with a C-SCI with an American Spinal Injury Association Impairment Scale (AIS) A or B score or an AIS C or D score, as well as in persons with a C3-C6 lesion or a C7-T1 lesion. The responsiveness of the VLT is significantly correlated to the GRT, but not to the FIM and the QIF. CONCLUSION: The VLT is responsive in measuring changes in AHSP during rehabilitation in persons with C-SCI.

A study on the interaction between hydroxylamine analogues and oxyhemoglobin in intact erythrocytes.

The oxidative potency of hydroxylamine (HYAM) and its O-derivatives (O-methyl- and O-ethyl hydroxylamine) is generally larger than the effects of the N-derivatives (N-methyl-, N-dimethyl-, and N,O-dimethyl hydroxylamine). The effects of the two groups of hydroxylamines also differ in a qualitative sense. To elucidate this difference in toxicity profiles we investigated the hemoglobin dependence of the toxicity, the occurrence of cell-damaging products like superoxide and H(2)O(2), and the cellular kinetics of the hydroxylamine analogues. All hydroxylamines were found to depend on the presence and accessibility of oxyhemoglobin to exert their toxicity. This did not provide an explanation for the different toxicity profiles. The interaction of some hydroxylamines with oxyhemoglobin is known to lead to the formation of radical intermediates. Differences in the stability of these radical products are known to occur, and in some cases secondary products are formed. This can contribute to the differences in toxicity. In this respect, production of superoxide radicals was demonstrated for all hydroxylamines in the reaction with oxyhemoglobin. Evidence for H(2)O(2) generation during the reaction of HYAM, O-methyl, O-ethyl-, and N-dimethyl hydroxylamine with oxyhemoglobin was also found. Next to variations in the products formed, differences in cellular kinetics are likely to be among the most important factors that explain the different toxicity patterns seen for the hydroxylamines in erythrocytes. Indeed, differences were found to exist for the kinetics of methemoglobin formation in erythrocytes. Not only was the final level of methemoglobin formed much lower for the N-derivatives, but also the reaction rate with oxyhemoglobin was slower than with HYAM and its O-derivatives. Except for N,O-dimethyl hydroxylamine (NODMH), the same pattern was seen in hemolysates. NODMH tripled its effect on hemoglobin in hemolysate compared with incubations in erythrocytes. This implies that cellular uptake is a limiting factor for NODMH. Since formation of H(2)O(2) is most likely a result of an interaction with hemoglobin, differences in kinetics of methemoglobin formation can be an explanation for the fact that NMH and NODMH did not produce H(2)O(2) to a detectable level. These results indicate that (a) the toxicity of all hydroxylamines depends on an interaction with oxyhemoglobin; (b) the interaction with hemoglobin produces radical intermediates and concomitantly superoxide radicals and H(2)O(2); and (c) differences in uptake, reaction rate with hemoglobin, and stability of the intermediates formed do exist for the different hydroxylamines and contribute to their differences in toxicity.

Only the glutathione dependent antioxidant enzymes are inhibited by haematotoxic hydroxylamines.

Hydroxylamine and some of its derivatives are known to cause oxidative effects both in vitro and in vivo. In the current study we investigated the effects of hydroxylamines on the enzymatic antioxidant defense system in human erythrocytes. The activity of catalase and superoxide dismutase was not significantly influenced by any of the hydroxylamines tested. However, the activity of glutathione peroxidase (GPX) and glutathione S-transferase (GST) was strongly inhibited by hydroxylamine and its O-derivatives (O-methyl and O-ethyl hydroxylamine). GPX was also inhibited by two N-derivatives of hydroxylamine (i.e. N-dimethyl and N,O-dimethyl hydroxylamine). This indicates that exposure to hydroxylamines not only changes the cellular oxidation-reduction status but also leads to inhibition of the glutathione dependent antioxidant enzymes. GST as well as GPX have cysteine residues at the active site of the enzymes. Such an accessible thiol group is generally susceptible to formation of protein-mixed disulphides or intramolecular disulphides. If these thiol groups are essential for activity this would be accompanied by an increase or decrease in the enzyme activity. In principle this is also true for glutathione reductase (GR), which in this study was only inhibited by N,O-dimethyl and N-methyl hydroxylamines. However, GR is capable to reduce these disulphides by taking up two electrons, either from its substrate NAPDH or from another reductant. Oxidation of these thiol groups in GR would thus not lead to impairment of GR activity. The fact that NODMH and NMH do decrease the GR activity can therefore only be explained by other modifications. The activity loss of GST and GPX on the other hand, is likely to involve oxidation of critical cysteine residues. The practical consequence of these findings is that the cellular prooxidant state that may arise in erythrocytes exposed to hydroxylamines can be further increased by activity loss of protective enzymes, which may decrease the average life span of the red blood cell.

Two mechanisms for toxic effects of hydroxylamines in human erythrocytes: involvement of free radicals and risk of potentiation.

The toxic potency of three industrially used hydroxylamines was studied in human blood cells in vitro. The parent compound hydroxylamine and the O-ethyl derivative gave very similar results. Both compounds induced a high degree of methemoglobin formation and glutathione depletion. Cytotoxicity was visible as Heinz body formation and hemolysis. High levels of lipid peroxidation occurred, in this respect O-ethyl hydroxylamine was more active than hydroxylamine. In contrast H2O2 induced lipid peroxidation was lowered after O-ethyl hydroxylamine or hydroxylamine treatment, this is explained by the ferrohemoglobin dependence of H2O2 induced radical species formation. Glutathione S-transferase (GST) and NADPH methemoglobin reductase (NADPH-HbR) activities were also impaired, probably as a result of the radical stress occurring. The riboflavin availability was decreased. Other enzyme activities glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH), glucose phosphate isomerase and NADH methemoglobin reductase, were not or only slightly impaired by hydroxylamine or O-ethyl hydroxylamine treatment. A different scheme of reactivity was found for N,O-dimethyl hydroxylamine. This compound gave much less methemoglobin formation and no hemolysis or Heinz body formation at concentrations up to and including 7 mM. Lipid peroxidase induction was not detectable, but could be induced by subsequent H2O2 treatment. GST and NADPH-HbR activities and riboflavin availability were not decreased. On the other hand GR and G6PDH activities were inhibited. These results combined with literature data indicate the existence of two different routes of hematotoxicity induced by hydroxylamines. Hydroxylamine as well as O-alkylated derivatives primarily induce methemoglobin, a process involving radical formation. The radical stress occurring is probably responsible for most other effects. N-alkylated species like N,O-dimethyl hydroxylamine primarily lead to inhibition of the protective enzymes G6PDH and GR. Since these enzymes play a key role in the protection of erythrocytes against oxidative stress a risk of potentiation during mixed exposure does exist.

In vitro haematotoxic effects of three methylated hydroxylamines.

Hydroxylamine (HYAM, HONH2) and some of its derivatives are known to cause erythrotoxic effects both in vitro and in vivo. Previous studies have shown that the primary in vitro effect of HYAM and O-ethyl hydroxylamine (OEH) is methaemoglobin formation, leading to liberation of free radicals which cause lipid peroxidation, enzyme inhibitions and glutathione depletion. By contrast, N-substituted N,O-dimethyl hydroxylamine (NODMH), primarily induces impairment of glucose 6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR). The oxidative potency of HYAM and the O-derivative was larger than the potency of the N,O-derivative. This seemed to indicate that attachment of an alkyl group to the nitrogen atom of hydroxylamine leads to decreased reactivity. To achieve a better understanding of the structure activity relationship for hydroxylamines three methylated derivatives were tested: N-methyl hydroxylamine (NMH). N-dimethyl hydroxylamine (NDMH) and O-methyl hydroxylamine (OMH). We were also interested in the erythrotoxic potency of OMH which recently entered industrial production. Methaemoglobin formation, high release of lipid peroxidation products, inhibition of NADPH methaemoglobin reductase and glutathione S-transferase (GST) and depletion of total glutathione (GT) were seen for OMH. The reducing enzymes G6PDH and GR were not impaired by OMH. These findings for OMH are consistent with the proposed mechanism for O-derivatives. Since both the effects caused by OMH and its potency are comparable to those of HYAM and OEH this indicates that possible occupational exposure to this compound may be approached similarly to HYAM and OEH. NMH only inhibited G6PDH and GR activity, which is fully in accord with the proposed mechanism for N-substituted derivatives of HYAM. However, NDMH a double N-substituted compound, caused a strikingly different scheme of reactivity inhibition of G6PDH but not of GR, severe methaemoglobin formation, only little lipid peroxidation and some impairment of NADPH methaemoglobin reductase. This study confirms that O-derivatives of HYAM are potent haemoglobin oxidators, leading to other oxidative effects. The main effect was confirmed for single N-derivatives as inhibition of the two protective enzymes G6PDH and GR. However, the results for NDMH indicate that this simple classification of O-derivatives and N-derivatives has to be extended for double N-substituted compounds which give a mixture of effects.

Hydroxylamine treatment increases glutathione-protein and protein-protein binding in human erythrocytes.

Hydroxylamine is a direct-acting hematotoxic agent leading to hemolytic anemia in animals and man. The effect of hydroxylamine on the morphology, sulfhydryl status and membrane skeletal proteins of human erythrocytes were studied. Loss of reduced glutathione (GSH) from the red blood cells was directly proportional to the hydroxylamine concentration used. This loss of GSH was larger than the sum of the increase in the amounts of extracellular glutathione and intracellular oxidized glutathione (GSSG). The extracellular glutathione is mainly present as GSSG, which is in agreement with the fact that only GSSG is exported from the erythrocytes by membrane bound ATPases. Lack of GSSG export was not limited by decreased ATP levels in the erythrocytes and we concluded that the GSH that disappeared did not become available as intracellular GSSG. After reduction of the erythrocyte incubates the lost GSH was almost completely recovered indicating that the lost GSH is present in the cell as protein-glutathione mixed disulfides. Glutathione thus stored within the cell can be quickly recovered by combined thioltransferase and glutathione reductase activity when conditions become more favorable again. SDS-polyacrylamide gel electrophoresis of membrane ghosts from human red cells revealed changes in skeletal proteins with a smearing of bands 1, 2 and 3 to the higher molecular weight end of the gel and the appearance of new monomeric and dimeric hemoglobin bands at about 16 and 30 kD. The observed alterations are probably a consequence of disulfide bridge formation between cellular proteins (mainly hemoglobin) and skeletal proteins as well as between hemoglobin monomers. Exposure of hydroxylamine to erythrocytes caused severe Heinz body formation but the outside morphology of the cells was only marginally altered. The described changes in sulfhydryl status of the red blood cells are likely to play a major role in the premature splenic sequestration of hydroxylamine-damaged erythrocytes.