Objectively measured activity is not associated with average pain intensity 1 week after surgery: A cross-sectional study.

BACKGROUND: Measures of physical activity and pain-related patient-reported outcomes are important components of patient recovery after surgery. However, little is known about their association in the early post-operative period. This study aims to increase this knowledge. Our primary objective was to determine the association between average pain intensity and activity (in steps) 1 week after surgery. Secondary objectives were the association of activity with other patient-reported outcomes, age, sex, comorbidities and body mass index. METHODS: Data were obtained from the PROMPT sub-project of IMI-PainCare. Patients after breast and endometriosis-related surgery, sternotomy and total knee arthroplasty completed pain-related outcomes questionnaires and wore an ActiGraph activity-tracking device. We correlated steps with average pain intensity on post-operative days 6 and 7. Secondary analyses were done using correlations and t-tests. RESULTS: In 284 cases, there was no statistically significant correlation between steps and average pain intensity. In addition, none of the 28 secondary analyses showed a statistically significant result. CONCLUSIONS: Pain-related patient-reported outcome measures and physical activity are separate entities. Both should be measured after surgery to assess patient recovery and to identify treatment deficiencies. SIGNIFICANCE STATEMENT: Measuring recovery is a multi-dimensional challenge. After surgery, clinicians need to be aware that neither pain intensity nor activity levels tell the whole story. Each can hint to problems and treatment requirements.

Spared Nerve Injury Causes Sexually Dimorphic Mechanical Allodynia and Differential Gene Expression in Spinal Cords and Dorsal Root Ganglia in Rats.

Neuropathic pain is more prevalent in women. However, females are under-represented in animal experiments, and the mechanisms of sex differences remain inadequately understood. We used the spared nerve injury (SNI) model in rats to characterize sex differences in pain behaviour, unbiased RNA-Seq and proteomics to study the mechanisms. Male and female rats were subjected to SNI- and sham-surgery. Mechanical and cold allodynia were assessed. Ipsilateral lumbar dorsal root ganglia (DRG) and spinal cord (SC) segments were collected for RNA-seq analysis with DESeq2 on Day 7. Cerebrospinal fluid (CSF) samples for proteomic analysis and DRGs and SCs for analysis of IB-4 and CGRP, and IBA1 and GFAP, respectively, were collected on Day 21. Females developed stronger mechanical allodynia. There were no differences between the sexes in CGRP and IB-4 in the DRG or glial cell markers in the SC. No CSF protein showed change following SNI. DRG and SC showed abundant changes in gene expression. Sexually dimorphic responses were found in genes related to T-cells (cd28, ctla4, cd274, cd4, prf1), other immunological responses (dpp4, c5a, cxcr2 and il1b), neuronal transmission (hrh3, thbs4, chrna4 and pdyn), plasticity (atf3, c1qc and reg3b), and others (bhlhe22, mcpt1l, trpv6). We observed significantly stronger mechanical allodynia in females and numerous sexually dimorphic changes in gene expression following SNI in rats. Several genes have previously been linked to NP, while some are novel. Our results suggest gene targets for further studies in the development of new, possibly sex-specific, therapies for NP.

Sensory profiles in women with neuropathic pain after breast cancer surgery.

PURPOSE: We performed a detailed analysis of sensory function in patients with chronic post-surgical neuropathic pain (NP) after breast cancer treatments by quantitative sensory testing (QST) with DFNS (German Research Network on Neuropathic Pain) protocol and bed side examination (BE). The nature of sensory changes in peripheral NP may reflect distinct pathophysiological backgrounds that can guide the treatment choices. NP with sensory gain (i.e., hyperesthesia, hyperalgesia, allodynia) has been shown to respond to Na+-channel blockers (e.g., oxcarbazepine). METHODS: 104 patients with at least "probable" NP in the surgical area were included. All patients had been treated for breast cancer 4-9 years ago and the handling of the intercostobrachial nerve (ICBN) was verified by the surgeon. QST was conducted at the site of NP in the surgical or nearby area and the corresponding contralateral area. BE covered the upper body and sensory abnormalities were marked on body maps and digitalized for area calculation. The outcomes of BE and QST were compared to assess the value of QST in the sensory examination of this patient group. RESULTS: Loss of function in both small and large fibers was a prominent feature in QST in the area of post-surgical NP. QST profiles did not differ between spared and resected ICBN. In BE, hypoesthesia on multiple modalities was highly prevalent. The presence of sensory gain in BE was associated with more intense pain. CONCLUSIONS: Extensive sensory loss is characteristic for chronic post-surgical NP several years after treatment for breast cancer. These patients are unlikely to respond to Na+-channel blockers.

Machine-learned selection of psychological questionnaire items relevant to the development of persistent pain after breast cancer surgery.

BACKGROUND: Prevention of persistent pain after breast cancer surgery, via early identification of patients at high risk, is a clinical need. Psychological factors are among the most consistently proposed predictive parameters for the development of persistent pain. However, repeated use of long psychological questionnaires in this context may be exhaustive for a patient and inconvenient in everyday clinical practice. METHODS: Supervised machine learning was used to create a short form of questionnaires that would provide the same predictive performance of pain persistence as the full questionnaires in a cohort of 1000 women followed up for 3 yr after breast cancer surgery. Machine-learned predictors were first trained with the full-item set of Beck's Depression Inventory (BDI), Spielberger's State-Trait Anxiety Inventory (STAI), and the State-Trait Anger Expression Inventory (STAXI-2). Subsequently, features were selected from the questionnaires to create predictors having a reduced set of items. RESULTS: A combined seven-item set of 10% of the original psychological questions from STAI and BDI, provided the same predictive performance parameters as the full questionnaires for the development of persistent postsurgical pain. The seven-item version offers a shorter and at least as accurate identification of women in whom pain persistence is unlikely (almost 95% negative predictive value). CONCLUSIONS: Using a data-driven machine-learning approach, a short list of seven items from BDI and STAI is proposed as a basis for a predictive tool for the persistence of pain after breast cancer surgery.

A machine-learned analysis of human gene polymorphisms modulating persisting pain points to major roles of neuroimmune processes.

BACKGROUND: Human genetic research has implicated functional variants of more than one hundred genes in the modulation of persisting pain. Artificial intelligence and machine-learning techniques may combine this knowledge with results of genetic research gathered in any context, which permits the identification of the key biological processes involved in chronic sensitization to pain. METHODS: Based on published evidence, a set of 110 genes carrying variants reported to be associated with modulation of the clinical phenotype of persisting pain in eight different clinical settings was submitted to unsupervised machine-learning aimed at functional clustering. Subsequently, a mathematically supported subset of genes, comprising those most consistently involved in persisting pain, was analysed by means of computational functional genomics in the Gene Ontology knowledgebase. RESULTS: Clustering of genes with evidence for a modulation of persisting pain elucidated a functionally heterogeneous set. The situation cleared when the focus was narrowed to a genetic modulation consistently observed throughout several clinical settings. On this basis, two groups of biological processes, the immune system and nitric oxide signalling, emerged as major players in sensitization to persisting pain, which is biologically highly plausible and in agreement with other lines of pain research. CONCLUSIONS: The present computational functional genomics-based approach provided a computational systems-biology perspective on chronic sensitization to pain. Human genetic control of persisting pain points to the immune system as a source of potential future targets for drugs directed against persisting pain. Contemporary machine-learned methods provide innovative approaches to knowledge discovery from previous evidence. SIGNIFICANCE: We show that knowledge discovery in genetic databases and contemporary machine-learned techniques can identify relevant biological processes involved in Persitent pain.

Ketamine and norketamine attenuate oxycodone tolerance markedly less than that of morphine: from behaviour to drug availability.

BACKGROUND: Ketamine attenuates morphine tolerance by antagonising N-methyl-d-aspartate receptors. However, a pharmacokinetic interaction between morphine and ketamine has also been suggested. The interaction between oxycodone and ketamine is unclear. We studied the effects of ketamine and norketamine on the attenuation of morphine and oxycodone tolerance focusing on both the pharmacodynamic and pharmacokinetic interactions. METHODS: Morphine 9.6 mg day-1 or oxycodone 3.6 mg day-1 was delivered to Sprague-Dawley rats by subcutaneous pumps. Once tolerance had developed, the rats received subcutaneous injections of ketamine or norketamine. Tail-flick, hot-plate, and rotarod tests were performed. Drug concentrations were measured with high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Anti-nociceptive tolerance to morphine and oxycodone developed similarly by Day 6. Acute ketamine 10 mg kg-1 and norketamine 30 mg kg-1 attenuated morphine tolerance for 120 and 150 min, respectively, whereas in oxycodone-tolerant rats the effect lasted only 60 min. Both ketamine and norketamine increased the brain and serum concentrations of morphine, and inhibited its metabolism to morphine-3-glucuronide, whereas oxycodone concentrations were not changed. Morphine, but not oxycodone, pretreatment increased the brain and serum concentrations of ketamine and norketamine. Ketamine, but not norketamine, significantly impaired the motor coordination. CONCLUSIONS: Ketamine and norketamine attenuated morphine tolerance more effectively than oxycodone tolerance. Ketamine and norketamine increased morphine, but not oxycodone brain concentrations, which may partly explain this difference. Norketamine is effective in attenuating morphine tolerance with minor effects on motor coordination. These results warrant pharmacokinetic studies in patients who are co-treated with ketamine and opioids.

Prediction of persistent post-surgery pain by preoperative cold pain sensitivity: biomarker development with machine-learning-derived analysis.

BACKGROUND: To prevent persistent post-surgery pain, early identification of patients at high risk is a clinical need. Supervised machine-learning techniques were used to test how accurately the patients' performance in a preoperatively performed tonic cold pain test could predict persistent post-surgery pain. METHODS: We analysed 763 patients from a cohort of 900 women who were treated for breast cancer, of whom 61 patients had developed signs of persistent pain during three yr of follow-up. Preoperatively, all patients underwent a cold pain test (immersion of the hand into a water bath at 2-4 °C). The patients rated the pain intensity using a numerical ratings scale (NRS) from 0 to 10. Supervised machine-learning techniques were used to construct a classifier that could predict patients at risk of persistent pain. RESULTS: Whether or not a patient rated the pain intensity at NRS=10 within less than 45 s during the cold water immersion test provided a negative predictive value of 94.4% to assign a patient to the "persistent pain" group. If NRS=10 was never reached during the cold test, the predictive value for not developing persistent pain was almost 97%. However, a low negative predictive value of 10% implied a high false positive rate. CONCLUSIONS: Results provide a robust exclusion of persistent pain in women with an accuracy of 94.4%. Moreover, results provide further support for the hypothesis that the endogenous pain inhibitory system may play an important role in the process of pain becoming persistent.

Pregabalin enhances the antinociceptive effect of oxycodone and morphine in thermal models of nociception in the rat without any pharmacokinetic interactions.

BACKGROUND: Oxycodone is increasingly being used in combination with pregabalin. Pregabalin use is prevalent in opioid-dependent individuals. A high number of deaths caused by the co-use of gabapentinoids and opioids occur. It is not known whether pregabalin affects concentrations of oxycodone or morphine in the central nervous system. METHODS: Effects of pregabalin on acute oxycodone or morphine-induced antinociception, tolerance and sedation were studied using tail-flick, hot plate and rotarod tests in male Sprague-Dawley rats. Concentrations of pregabalin, opioids and their major metabolites in the brain were quantified by mass spectrometry. RESULTS: In the hot plate test, morphine (2.5 mg/kg, s.c.) caused antinociception of 28% maximum possible effect (MPE), whereas pregabalin (50 mg/kg, i.p.) produced 8-10% MPE. Co-administration of pregabalin and morphine resulted in antinociception of 63% MPE. Oxycodone (0.6 mg/kg s.c.) produced antinociception of 18% MPE, which increased to 39% MPE after co-administration with pregabalin. When pregabalin 10 mg/kg was administered before oxycodone (0.6 mg/kg, s.c.) or morphine (2.5 mg/kg), only the effect of oxycodone was potentiated in the tail-flick and the hot plate tests. Brain concentrations of the opioids, their major metabolites and pregabalin were unchanged. Pregabalin co-administration (50 mg/kg, i.p., once daily) did not prevent the development of morphine tolerance. CONCLUSIONS: Pregabalin potentiated antinociceptive and sedative effects of oxycodone and morphine in acute nociception. Co-administration of pregabalin with the opioids did not affect the brain concentrations of oxycodone or morphine. Pregabalin did not prevent morphine tolerance.

Patients with complex regional pain syndrome overestimate applied force in observed hand actions.

BACKGROUND: Movement accuracy is ensured by interaction between motor, somatosensory, and visual systems. In complex regional pain syndrome (CRPS), this interaction is disturbed. To explore CRPS patients' visual perception of actions, we investigated how these patients evaluate the applied force in observed hand actions of another person. METHODS: Nineteen patients suffering from unilateral upper-limb CRPS and 19 healthy control subjects viewed six different videos of left- and right-hand actions. They were asked to evaluate the applied force in each hand action, as well as their subjective sensations of unpleasantness and pain during the observation. RESULTS: The patients overestimated the force applied in the videos: the ratings were two times as large as in the control subjects for actions performed with the hand corresponding to the patients' affected hand, and 1.5 times as large for actions corresponding to their healthy hand. The control subjects considered the stimuli neutral and painless, whereas the patients rated them unpleasant. Moreover, the patients felt increased pain during viewing actions performed with the hand corresponding to their affected side. The overestimation of force was related to the elicited unpleasantness and pain, but not to the patients' muscle strength. CONCLUSIONS: We propose that the overestimation of force is explained both by the pain elicited by the observation and by the abnormal sensorimotor integration that is associated with perception of increased effort. This visually elicited unpleasantness and painfulness may promote avoidance of viewing own actions, further impairing the patients' motor performance.

Ketamine coadministration attenuates morphine tolerance and leads to increased brain concentrations of both drugs in the rat.

BACKGROUND AND PURPOSE: The effects of ketamine in attenuating morphine tolerance have been suggested to result from a pharmacodynamic interaction. We studied whether ketamine might increase brain morphine concentrations in acute coadministration, in morphine tolerance and morphine withdrawal. EXPERIMENTAL APPROACH: Morphine minipumps (6 mg·day(-1) ) induced tolerance during 5 days in Sprague-Dawley rats, after which s.c. ketamine (10 mg·kg(-1) ) was administered. Tail flick, hot plate and rotarod tests were used for behavioural testing. Serum levels and whole tissue brain and liver concentrations of morphine, morphine-3-glucuronide, ketamine and norketamine were measured using HPLC-tandem mass spectrometry. KEY RESULTS: In morphine-naïve rats, ketamine caused no antinociception whereas in morphine-tolerant rats there was significant antinociception (57% maximum possible effect in the tail flick test 90 min after administration) lasting up to 150 min. In the brain of morphine-tolerant ketamine-treated rats, the morphine, ketamine and norketamine concentrations were 2.1-, 1.4- and 3.4-fold, respectively, compared with the rats treated with morphine or ketamine only. In the liver of morphine-tolerant ketamine-treated rats, ketamine concentration was sixfold compared with morphine-naïve rats. After a 2 day morphine withdrawal period, smaller but parallel concentration changes were observed. In acute coadministration, ketamine increased the brain morphine concentration by 20%, but no increase in ketamine concentrations or increased antinociception was observed. CONCLUSIONS AND IMPLICATIONS: The ability of ketamine to induce antinociception in rats made tolerant to morphine may also be due to increased brain concentrations of morphine, ketamine and norketamine. The relevance of these findings needs to be assessed in humans.

Normal laser-evoked cortical responses in patients with chronic hemibody pain.

BACKGROUND: Patients with widespread unilateral chronic pain associated with recurrent herpes simplex virus (HSV) infections show functional and/or structural changes in the insula, anterior cingulate cortex, frontal and prefrontal cortices, as well as the thalamus, suggesting central dysfunction of the pain system in these patients. Central pain has been associated with attenuated laser-evoked cortical responses. We aimed to clarify whether the observed deficient activation of these areas to acute nociceptive stimuli is due to a lesion at a lower level of pain processing pathways. METHODS: We explored the functional integrity of the ascending nociceptive pathways by recording the cortical-evoked responses to noxious laser stimulation using magnetoencephalography and electroencephalography in eight patients (age 41-51 years, mean 46) with recurrent HSV infections and a history of chronic, spontaneous, widespread unilateral pain, and in nine age-matched healthy control subjects. RESULTS: The cortical-evoked fields of the HSV patients originating from the secondary somatosensory and posterior parietal cortices, as well as the evoked potentials recorded from the midline, did not differ from those of the control subjects, indicating functionally intact ascending nociceptive pathways. CONCLUSIONS: The present results show that our patients with chronic hemibody pain do not show signs of spinothalamic tract lesion. This indicates normal processing of sensory aspects of painful stimuli, while higher pain processing areas show altered activation. We conclude that normal laser-evoked magnetic fields (LEF) or laser-evoked potentials (LEP) may not exclude central pain condition.

Same-day and delayed reports of pain intensity in second-trimester medical termination of pregnancy: a brief report.

OBJECTIVE: To assess same-day and delayed reports of pain intensity during and after second-trimester medical termination of pregnancy (MTOP). STUDY DESIGN: A prospective randomized trial (217 women) comparing 1- and 2-day mifepristone-misoprostol intervals. RESULTS: Women reported intense pain [median visual analogue scale (interquartile range)] related to expulsion of the fetus [6 (0-10)]. Delayed reports of maximal pain described the pain as more intense than same-day reports [8 (3-10) vs. 7 (1-10), p<.001]. CONCLUSIONS: Most women reported and readily remembered intense pain associated with fetal expulsion during second-trimester MTOP. IMPLICATIONS: Adequate, properly timed pain management during second-trimester MTOP is crucial.

The mineralocorticoid receptor antagonist spironolactone enhances morphine antinociception.

BACKGROUND: Spironolactone, a commonly used mineralocorticoid receptor antagonist, has been reported to potentiate the effect of morphine in the rat. The aim of this study was to investigate the effects of spironolactone on morphine antinociception and tissue distribution. METHODS: The effects of spironolactone on acute morphine-induced antinociception, induction of morphine tolerance and established morphine tolerance were studied with tail-flick and hot plate tests in male Sprague-Dawley rats. Serum, brain, and liver morphine and its metabolite concentrations were quantified using high-pressure liquid chromatography-tandem mass spectrometry. Spironolactone was also administered with the peripherally acting, P-glycoprotein (P-gp) substrate loperamide to test whether spironolactone allows loperamide to pass the blood-brain barrier. RESULTS: Spironolactone (50 mg/kg, i.p.) had no antinociceptive effects of its own, but it enhanced the antinociceptive effect of morphine in both thermal tests. Two doses of spironolactone enhanced the maximum possible effect (MPE) from 19.5% to 100% in the hot plate test 90 min after administration of 4 mg/kg morphine. Morphine concentrations in the brain were increased fourfold at 90 min by spironolactone. Spironolactone did not inhibit the formation of morphine-3-glucuronide. Acute spironolactone restored morphine antinociception in morphine-tolerant rats but did not inhibit the development of tolerance. The peripherally restricted opioid, loperamide (10 mg/kg), had no antinociceptive effects when administered alone, but co-administration with spironolactone produced a 40% MPE in the hot plate test. CONCLUSIONS: Spironolactone has no antinociceptive effects in thermal models of pain, but it enhances the antinociceptive effects of morphine mainly by increasing morphine central nervous system concentrations, probably by inhibiting P-gp.

Transforming pain medicine: adapting to science and society.

The field of chronic pain medicine is currently facing enormous challenges. The incidence of chronic pain is increasing worldwide, particularly in the developed world. As a result, chronic pain is imposing a growing burden on Western societies in terms of cost of medical care and lost productivity. This burden is exacerbated by the fact that despite research efforts and a huge expenditure on treatment for chronic pain, clinicians have no highly effective treatments or definitive diagnostic measures for patients. The lack of an objective measure for pain impedes basic research into the biological and psychological mechanisms of chronic pain and clinical research into treatment efficacy. The development of objective measurements of pain and ability to predict treatment responses in the individual patient is critical to improving pain management. Finally, pain medicine must embrace the development of a new evidence-based therapeutic model that recognizes the highly individual nature of responsiveness to pain treatments, integrates bio-psycho-behavioural approaches, and requires proof of clinical effectiveness for the various treatments we offer our patients. In the long-term these approaches will contribute to providing better diagnoses and more effective treatments to lessen the current challenges in pain medicine.

Development of a screening instrument for risk factors of persistent pain after breast cancer surgery.

BACKGROUND: Persistent postsurgical pain can have a significant effect on the quality of life of women being treated for breast cancer. The aim of this prospective study was to develop a screening tool to identify presurgical demographic, psychological and treatment-related factors that predict persistence of significant pain in the operated area after 6 months from surgery. METHODS: Background and self-reported questionnaire data were collected the day before surgery and combined with treatment-related data. Pain in the operated area was assessed 6 months after surgery with a questionnaire. The Bayesian model was used for the development of a screening tool. RESULTS: Report of preoperative chronic pain, more than four or more previous operations, preoperative pain in the area to be operated, high body mass index, previous smoking and older age were included in the six-factor model that best predicted significant pain at the follow-up in the 489 women studied. CONCLUSION: A six-factor risk index was developed to estimate the risk of developing significant pain after breast cancer surgery. Neither treatment- nor mood-related variables were included in the model. Identification of risk factors may lead to prevention of persistent postsurgery pain. This tool could be used for target prevention to those who are at the highest risk of developing persistent postsurgery pain.

Effect of pain on autonomic nervous system indices derived from photoplethysmography in healthy volunteers.

BACKGROUND: Photoplethysmographic pulse wave amplitude (PPGA) and heart rate (HR) can be used to measure cold, nociception-induced autonomic responses, or both. The aim of our study was to correlate the intensity of experimental pain to changes in physiological variables reflecting the autonomic nervous system response to pain. METHODS: PPGA, HR, and subjective measurements of pain intensity were measured in 29 healthy male volunteers during two heat stimuli (43°C and 48°C) and the cold pressor test (CPT). Surgical pleth index (SPI), autonomic nervous system state (ANSS), and ANSS index (ANSSi) were calculated using PPGA and HR. RESULTS: Pain intensity scores increased on the average by 1.6, 3.5, and 8.1 for the 43°C, 48°C, and CPT stimuli, respectively. The pain intensity scores for all three stimuli groups were significantly different from each other (P<0.001). All three stimuli changed HR, PPGA, SPI, ANSS, and ANSSi values significantly from their respective baseline values (P<0.001 for all). Heat stimuli-induced pain intensity did not correlate with the magnitude of the respective changes in HR, PPGA, SPI, ANSS, and ANSSi. CPT-induced pain intensity correlated with the magnitude of the respective changes in HR, PPGA, SPI, ANSS, and ANSSi. PPGA, ANSSi, ANSS, and SPI differentiated between heat and cold stimuli-induced pain. CONCLUSIONS: All three thermal stimuli produced a significant change in photoplethysmograph-derived parameters. All photoplethysmograph-derived parameters appear to be suitable to study autonomic nervous system activation.

Inhibitors of catechol-O-methyltransferase sensitize mice to pain.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) inhibitors are used in Parkinson's disease in which pain is an important symptom. COMT polymorphisms modulate pain and opioid analgesia in humans. In rats, COMT inhibitors have been shown to be pro-nociceptive in acute pain models, but also to attenuate allodynia and hyperalgesia in a model of diabetic neuropathy. Here, we have assessed the effects of acute and repeated administrations of COMT inhibitors on mechanical, thermal and carrageenan-induced nociception in male mice. EXPERIMENTAL APPROACH: We used single and repeated administration of a peripherally restricted, short-acting (nitecapone) and also a centrally acting (3,5-dinitrocatechol, OR-486) COMT inhibitor. We also tested CGP 28014, an indirect inhibitor of COMT enzyme. Effects of OR-486 on thermal nociception were also studied in COMT deficient mice. Effects on spinal pathways were assessed in rats given intrathecal nitecapone. KEY RESULTS: After single administration, both nitecapone and OR-486 reduced mechanical nociceptive thresholds and thermal nociceptive latencies (hot plate test) at 2 and 3 h, regardless of their brain penetration. These effects were still present after chronic treatment with COMT inhibitors for 5 days. Intraplantar injection of carrageenan reduced nociceptive latencies and both COMT inhibitors potentiated this reduction without modifying inflammation. CGP 28014 shortened paw flick latencies. OR-486 did not modify hot plate times in Comt gene deficient mice. Intrathecal nitecapone modified neither thermal nor mechanical nociception. CONCLUSIONS AND IMPLICATIONS: Pro-nociceptive effects of COMT inhibitors were confirmed. The pro-nociceptive effects were primarily mediated via mechanisms acting outside the brain and spinal cord. COMT protein was required for these actions.