Inferior ectopic bone formation of mesenchymal stromal cells from adipose tissue compared to bone marrow: rescue by chondrogenic pre-induction.

Human mesenchymal stromal cells derived from bone marrow (BMSC) and adipose tissue (ATSC) represent a valuable source of progenitor cells for cell therapy and tissue engineering. While ectopic bone formation is a standard activity of human BMSC on calcium phosphate ceramics, the bone formation capacity of human ATSC has so far been unclear. The objectives of this study were to assess the therapeutic potency of ATSC for bone formation in an ectopic mouse model and determine molecular differences by standardized comparison with BMSC. Although ATSC contained less CD146(+) cells, exhibited better proliferation and displayed similar alkaline phosphatase activity upon osteogenic in vitro differentiation, cells did not develop into bone-depositing osteoblasts on ß-TCP after 8weeks in vivo. Additionally, ATSC expressed less BMP-2, BMP-4, VEGF, angiopoietin and IL-6 and more adiponectin mRNA, altogether suggesting insufficient osteochondral commitment and reduced proangiogenic activity. Chondrogenic pre-induction of ATSC/ß-TCP constructs with TGF-ß and BMP-6 initiated ectopic bone formation in >75% of samples. Both chondrogenic pre-induction and the osteoconductive microenvironment of ß-TCP were necessary for ectopic bone formation by ATSC pointing towards a need for inductive conditions/biomaterials to make this more easily accessible cell source attractive for future applications in bone regeneration.

Perioperative use of continuous renal replacement therapy for orthotopic liver transplantation.

PURPOSE: We present a retrospective study describing the perioperative use of continuous renal replacement therapy (CRRT) for orthotopic liver transplantation (OLT). MATERIALS AND METHODS: We retrospectively reviewed the clinical course of patients who underwent OLT with the perioperative use of CRRT. The following variables were recorded: Gender, age, indication for transplantation, time when CRRT was initiated, postoperative need for CRRT, and the patient and organ (liver, kidneys) outcome up to 1 year after transplantation. RESULTS: Among 105 patients who underwent OLT from 2006 to 2010; we used CRRT in 12 cases (11.4%) perioperatively, including 9 (8.3%) patients intraoperatively. Perioperative CRRT was employed for volume, electrolyte, and/or pH management. All patients who underwent CRRT perioperatively were alive at 1 month, 10 (83.3%), at 3 and 6 months and 9 (75%) at 1 year after OLT. Only 1 surviving patient (8.3%) required renal replacement therapy at 1 month after surgery. Renal replacement therapy was not required in any surviving patient up to 12 months posttransplantation. CONCLUSION: Perioperative and especially intraoperative use of CRRT therapy can potentially improve the outcomes of patients undergoing OLT.

Genetic determinants of platelet reactivity during acetylsalicylic acid therapy in diabetic patients: evaluation of 27 polymorphisms within candidate genes.

AIMS: Decreased platelet responsiveness to acetylsalicylic acid (ASA) reported previously in diabetic patients could be attributed to patient-based, clinical, genetic and cellular factors. The objective of the present study was to investigate the effect of the genomic polymorphism on the platelet reactivity in diabetic patients treated with ASA. METHODS AND RESULTS: The study cohort consisted of 295 Caucasians with diabetes type 2 who had been taking ASA tablets at the dose of 75 mg per day for at least 3 months for primary or secondary prevention of myocardial infarction (MI). Platelet reactivity analyzes were performed using VerifyNow ASA and PFA-100 assays. Genotyping for the selected 27 single nucleotide polymorphisms (SNPs) within 19 genes was performed using a Sequenom iPLEX platform. The results indicate that the statistically significant differences in platelet reactivity were observed in the PFA-100 assay for SNPs in following genes: TXBA2R (rs1131882), ADRA2A (rs4311994), PLA2G7 (rs7756935) and 9p21.3 (rs10120688) (P = 0.02, P = 0.03, P = 0.02, P = 0.03, respectively, all significance levels corrected for multiple comparisons). When using the VerifyNow ASA test, a weak nominal statistical significance (i.e. before multiple comparison testing) was observed for two SNPs in the GPVI gene: rs1671152 and rs1613662 [P = 0.025 (0.5) for both SNPs, corrected for multiple comparisons test]. CONCLUSIONS: The results from the present study suggest that the four analyzed genes may contribute to platelet reactivity measured with the PFA-100 assay in the diabetic population treated with ASA.

Prediction of in vivo bone forming potency of bone marrow-derived human mesenchymal stem cells.

Human mesenchymal stem cells (MSC) have attracted much attention for tissue regeneration including repair of non-healing bone defects. Heterogeneity of MSC cultures and considerable donor variability however, still preclude standardised production of MSC and point on functional deficits for some human MSC populations. We aimed to identify functional correlates of donor-dependency of bone formation in order to develop a potency assay predicting the therapeutic capacity of human MSC before clinical transplantation. MSC from 29 donors were characterised in vitro and results were correlated to bone formation potency in a beta-tricalcium-phosphate (ß-TCP)-scaffold after subcutaneous implantation into immunocompromised mice. In contrast to osteogenic in vitro differentiation parameters, a doubling time below 43.23 hours allowed to predict ectopic bone formation at high sensitivity (81.8%) and specificity (100%). Enriched conditions adapted from embryonic stem cell expansion rescued bone formation of inferior MSC populations while growth arrest of potent MSC by mitomycin C abolished bone formation, establishing a causal relationship between neo-bone formation and growth. Gene expression profiling confirmed a key role for proliferation status for the bone forming ability suggesting that a rate limiting anabolism and open chromatin determined and predicted the therapeutic potency of culture-expanded MSC. Proliferation-based potency testing and switch to enriched expansion conditions may pave the way for standardised production of MSC for bone repair.

Fatal hyperammonemia after renal transplant due to late-onset urea cycle deficiency: a case report.

We present a case of severe hyperammonemia with subsequent brain herniation in an adult man after renal transplantation. After successful surgery and an initially uneventful postoperative course, the patient developed significant mental status changes associated with seizure activity. His condition rapidly deteriorated, requiring mechanical ventilation and cardiovascular support. Laboratory studies at that time demonstrated an increased serum ammonia level without evidence of liver or kidney dysfunction. Further investigation revealed an increased orotic acid level in the urine, suggesting a urea cycle disorder (UCD). Despite aggressive therapy, the patient's condition continued to deteriorate. Magnetic resonance imaging demonstrated severe brain edema with no cerebral perfusion; after consultation with the family, care was withdrawn. The combination of hyperammonemia and elevated urine orotic acid with normal liver and kidney function suggested a UCD. It is important to note that patients with a UCD may be free of symptoms for many years. Several factors are able to trigger the disease in adulthood, leading to encephalopathy and death. In this case, the patient's seizures were initially assumed to be a side effect of immunosuppressive therapy. Further diagnostic measures were only performed late in the course of the disease, which delayed the diagnosis of UCD.

Comparison of two different temperature maintenance strategies during open abdominal surgery: upper body forced-air warming versus whole body water garment.

BACKGROUND: A new system has been developed that circulates warm water through a whole body garment worn by the patient during surgery. In this study the authors compared two different strategies for the maintenance of intraoperative normothermia. One strategy used a new water garment warming system that permitted active warming of both the upper and lower extremities and the back. The other strategy used a single (upper body) forced-air warming system. METHODS: In this prospective, randomized study, 53 adult patients were enrolled in one of two intraoperative temperature management groups during open abdominal surgery with general anesthesia. The water-garment group (n = 25) received warming with a body temperature (rectal) set point of 36.8 degrees C. The forced-air-warmer group (n = 28) received routine warming therapy using upper body forced-air warming system (set on high). The ambient temperature in the operating room was maintained constant at approximately 20 degrees C. Rectal, distal esophageal, tympanic, forearm, and fingertip temperatures were recorded perioperatively and during 2 h after surgery. Extubated patients in both groups were assessed postoperatively for shivering, use of additional warming devices, and subjective thermal comfort. RESULTS: The mean rectal and esophageal temperatures at incision, 1 h after incision, at skin closure, and immediately postoperatively were significantly higher (0.4-0.6 degrees C) in the group that received water-garment warming when compared with the group that received upper body forced-air warming. The calculated 95% confidence intervals for the above differences in core temperatures were 0.7-0.1, 0.8-0.2, 0.8-0.2, and 0.9-0.1, retrospectively. In addition, 14 and 7% of patients in the control upper body forced-air group remained hypothermic (< 35.5 degrees C) 1 and 2 h after surgery, respectively. No core temperature less than 35.5 degrees C was observed perioperatively in any of the patients from the water-garment group. A similar frequency of the thermal stress events (shivering, use of additional warming devices, subjective thermal discomfort) was observed after extubation in both groups during the 2 h after surgery. CONCLUSIONS: The investigated water warming system, by virtue of its ability to deliver heat to a greater percentage of the body, results in better maintenance of intraoperative normothermia that does forced-air warming applied only to the upper extremities, as is common practice.

Interspecies differences in hepatic Ca(2+)-ATPase activity and the effect of cold preservation on porcine liver Ca(2+)-ATPase function.

The accumulation of intracellular calcium ([Ca(2+)](i)) caused by ischemia-reperfusion during liver transplantation has been implicated as a factor leading to primary graft nonfunction. Plasma membrane (PM) and endoplasmic reticulum (ER) Ca(2+)-adenosinetriphosphatases (ATPases) are the primary transporters that maintain [Ca(2+)](i) homeostasis in the liver. We hypothesized that the porcine liver is better than the rat liver as a model for the study of human liver Ca(2+)-ATPase activity. We also hypothesized that cold preservation would depress Ca(2+)-ATPase activity in the porcine liver. Pig and rat livers were harvested, and human liver samples were obtained from surgical resection specimens. All were preserved with University of Wisconsin solution, and porcine livers were also preserved on ice for 2 to 18 hours. Ca(2+)-ATPase activity was measured after incubation with (45)Ca(2+) and adenosine triphosphate in the presence of specific Ca(2+)-ATPase inhibitors. Porcine PM and ER Ca(2+)-ATPase activities were 0.47 +/- 0.03 and 1.57 +/- 0.10 nmol of Ca(2+)/mg of protein/min, respectively. This was not significantly different from human liver, whereas rat liver was significantly greater at 2.60 +/- 0.03 and 9.2 +/- 0.9 nmol of Ca(2+)/mg of protein/min, respectively. We conclude that the Ca(2+)-ATPase activity in the pig liver is equivalent to that of human liver, and thus, the pig liver is a better model than the rat liver. Cold preservation studies showed a significant decrease in porcine hepatic PM Ca(2+)-ATPase activity after 4 hours of storage and near-total inhibition after 12 hours. Porcine hepatic ER Ca(2+)-ATPase activity showed a 45% decrease in activity by 12 hours and a 69% decrease by 18 hours. We conclude that cold ischemia at clinically relevant times depresses PM Ca(2+)-ATPase more than ER Ca(2+)-ATPase activity in pig liver homogenates.

Intravenous chloroprocaine attenuates hemodynamic changes associated with direct laryngoscopy and tracheal intubation.

We compared the effects of an IV administration of chloroprocaine and lidocaine on circulatory responses associated with endotracheal intubation. Thirty patients were randomly allocated to receive normal saline (placebo), lidocaine (1.5 mg/kg), or preservative-free chloroprocaine (4.5 mg/kg) 45 s before endotracheal intubation. Blood pressures and heart rate and rhythm were recorded before laryngoscopy and at 0.5, 1, 1.5, 2, 3, and 5 min after intubation. Blood samples were analyzed for catecholamine and chloroprocaine concentrations. Chloroprocaine reduced increases in blood pressure in response to intubation when compared with patients receiving normal saline and lidocaine. Systolic blood pressures at 0.5 and 1 min after intubation were significantly lower in the chloroprocaine group when compared with both the control and lidocaine groups (P < 0.05). Diastolic and mean blood pressures were significantly lower in the chloroprocaine group at all time points until 5 min after intubation (P < 0.05). Chloroprocaine and, to a lesser degree, lidocaine, produced marked attenuation of intubation-induced increases in plasma concentration of epinephrine and norepinephrine. Plasma concentrations of norepinephrine were significantly smaller in the chloroprocaine group at 0.5, 1, and 1.5 min, and plasma concentrations of epinephrine were significantly smaller at 0.5 after intubation when compared with control and lidocaine groups (P < 0.05). Measurable concentrations of chloroprocaine were recorded in plasma samples for 2 min after its administration. No adverse chloroprocaine effects (i.e., circulatory disturbances, venous irritation) were detected. The IV administration of chloroprocaine effectively blunted cardiovascular response produced by laryngoscopy and endotracheal intubation, and this effect was more pronounced when compared with IV lidocaine.

Lack of effect of flurothyl, a non-anesthetic fluorinated ether, on rat brain synaptic plasma membrane calcium-ATPase.

Plasma membrane Ca2+-ATPase (PMCA), a regulator of intracellular calcium, is inhibited by volatile anesthetics and by xenon and nitrous oxide. Response of a cellular system to anesthetics, particularly to volatile agents, raises the question of non-specific, even toxic, side effects unrelated to anesthetic action. Compounds with chemical and physical properties similar to halogenated anesthetics, but which lack anesthetic effect, have been used to address this question. We have compared the effects of halothane and flurothyl, a non-anesthetic fluorinated ether, on PMCA Ca2+ transport across isolated brain synaptic plasma membranes (SPM). Flurothyl, at concentrations predicted by the Meyer-Overton curve to range from 0.4 to 2.6 MAC (minimum alveolar concentration), had no significant on PMCA activity. In contrast halothane, 1.3 MAC, reduced Ca2+ transport 30 to 40%. These findings provide further evidence for a specific effect of inhalation anesthetics on neuronal plasma membrane Ca2+-ATPase.

Anesthetic-induced alteration of Ca2+ homeostasis in neural cells: a temperature-sensitive process that is enhanced by blockade of plasma membrane Ca2+-ATPase isoforms.

BACKGROUND: Many inhalation anesthetics at clinically relevant concentrations inhibit plasma membrane Ca2+-adenosine triphosphatase (PMCA) ion pumping in brain synaptic membranes and in cultured cells of neural origin. In this study, the authors investigated the effect of inhalation anesthetics on cytosolic calcium homeostasis in cortical neurons maintained at physiologic and room temperatures and on cortical neurons and pheochromocytoma cells with antisense blockade of specific PMCA isoforms. METHODS: Using Ca2+-specific confocal microfluorimetry, the anesthetic effects on Ca2+ dynamics were examined in mouse embryonic cortical neurons in association with ligand-stimulated Ca2+ influx. Studies were done at 21 degrees C and 37 degrees C. Mouse embryonic cortical neurons with oligodeoxyribonucleotide blockade of PMCA2 expression and transfected rat pheochromocytoma cells with blocked expression of PMCA1 were also examined. RESULTS: Baseline and poststimulation peak cytosolic calcium concentrations ([Ca2+]i) were increased, and Ca2+ clearance was delayed in cells exposed at 37 degrees C, but not at 21 degrees C, to concentrations < or = 1 minimum alveolar concentration (MAC)-equivalent of halothane, isoflurane, and sevoflurane. Neurons exposed to xenon solutions < or = 0.4, 0.6, and 0.8 MAC showed dose-related perturbations of cytosolic Ca2+. Calcium dynamics were altered in neural cells with blocked PMCA isoform production, but at much lower halothane concentrations: 0.5 MAC for cortical neurons and 0.1 MAC for pheochromocytoma cells. CONCLUSIONS: By extruding Ca2+ through the plasma membrane, PMCA maintains resting neuronal [Ca2+]i at low levels and clears physiologic loads of Ca2+ after influx through calcium channels. Inhalation anesthetics perturb this process and thus may interfere with neurotransmitter release, altering interneuronal signaling.

Decreased patient analgesic requirements after liver transplantation and associated neuropeptide levels.

BACKGROUND: Decreased morphine requirements have been reported after liver transplantation when compared with other types of major abdominal surgery. The aim of this study was to examine plasma concentrations of three neuropeptides involved in pain modulation-metenkephalin (ME), beta-endorphin (BE), and substance P (SP)-in patients undergoing orthotopic liver transplantation (OLT) and in control patients undergoing other liver operations. We then compared the postoperative analgesic requirements in these two groups of patients. METHODS: Plasma levels of ME, BE, and SP were measured by radioimmunoassay at preincision, preemergence, and for 3 days after operation in 13 patients undergoing OLT and in 10 control patients. Patient-controlled analgesia morphine delivery was recorded for all patients postoperatively, and plasma morphine, its metabolites, and patient pain and sedation scores were also measured. RESULTS: ME levels were elevated in all OLT patient samples when compared with control patient samples. BE levels were not significantly different at any time. SP levels were significantly decreased only in preincision and preemergence OLT patient samples. Total patient-controlled analgesia morphine delivered during the first 3 postoperative days was significantly less in OLT patients (70+/-8 mg) than in control patients (101+/-12 mg). Plasma morphine, morphine-3-glucuronide, and morphine-6-glucuronide levels were decreased in OLT patients, however, statistical significance was seen only in the morphine-6-glucuronide results. CONCLUSIONS: We have shown that postoperative analgesic requirements are decreased in OLT patients, and we suggest that associated increased peripheral ME levels may be contributing to this decreased requirement. Based on our results, circulating BE and SP are less significant factors affecting postoperative analgesic requirements.

Hepatic energy charge and adenine nucleotide status in rats anesthetized with halothane, isoflurane or enflurane.

BACKGROUND: Volatile anesthetics are known to have varying effects on hepatic oxygen supply in vivo and have been shown to depress hepatic mitochondrial respiration and so energy charge in vitro. However, the effect of halothane, isoflurane and enflurane on hepatic adenine nucleotide status in vivo has not been evaluated. METHODS: Ninety male rats were exposed to 40% oxygen (n = 22) or 40% oxygen in equipotent (1 MAC) concentrations of halothane (1%) (n = 23), isoflurane (1.4%) (n = 22) or enflurane (2%) (n = 23) for 2 hours. All animals were then administered intraperitoneal pentobarbital and anesthesia continued and laparotomy was performed. A liver biopsy was taken for determination of hepatocellular adenosine-5-triphosphate (ATP), adenosine-5-diphosphate (ADP) and adenosine-5-monophosphate (AMP) and computation of energy charge (EC) from ¿(ATP + 1/2ADP)+(ATP + ADP + AMP)¿ and total adenine nucleotides (TAN) from (ATP + ADP + AMP). After the biopsy the aorta was cannulated for blood sampling. RESULTS: Rats in each group were similar in weight, as well as acid base and blood gas status just after liver biopsy. Hepatic energy charge, ATP, ADP, AMP, and TAN levels were not different in animals receiving either halothane, isoflurane or enflurane when compared with those receiving only oxygen. CONCLUSION: One MAC of anesthesia for a period of 2 hours with the described volatile anesthetic agents did not affect adenine nucleotide status in vivo in rats.

Liver transplantation is associated with increased met-enkephalin levels in the pig.

BACKGROUND: It has been reported that less postoperative morphine is required following liver transplantation than is required following open cholecystectomy. This may be attributable to endogenous factors rather than to altered morphine pharmacokinetics. We measured the plasma concentrations of two endogenous neuropeptides associated with pain modulation, substance P (SP) and met-enkephalin (ME), in pigs undergoing liver transplantation and in control pigs undergoing laparotomy. METHODS: With the approval of the institutional Animal Care Committee, pigs were anesthetized with ketamine (30 mg/ kg,i.m.), atropine (0.05 mg/kg, i.m.) and acetylpromazine (0.1 mg/kg, i.m.). Anesthesia was maintained with isoflurane in oxygen. Pigs in the transplantation group (n = 10) underwent liver transplantation and control pigs (n = 10) underwent laparotomy. Blood samples for SP and ME measurement were collected pre-incision (Pre-In), pre-emergence (Pre-Em) from anesthesia, 6-12 hours, 18 hours, and 24 hours after surgery. SP and ME levels were determined by radioimmunoassay. Results are expressed as mean +/- SEM (in pg/ml of plasma for both peptides) and were compared by the non-parametric Mann-Whitney U test. Statistical significance was inferred if P < 0.05. RESULTS: Plasma ME levels were significantly increased in the transplanted pigs at Pre-Em, 6-12 hours and 18 hours after surgery. No statistically significant difference was observed for plasma SP level between the control and transplant pigs. CONCLUSIONS: Liver transplantation in the pig model is associated with increased concentrations of endogenous ME (but not SP) in plasma for at least 18 hours after surgery as compared to animals undergoing laparotomy.

Halothane alters electrical activity and calcium dynamics in cultured mouse cortical, spinal cord, and dorsal root ganglion neurons.

Halothane inhibits neural plasma membrane Ca(2+)-ATPase, a pump that ejects Ca2+ from the cell after influx through voltage- or ligand-activated channels. Intracellular microelectrode recordings in mouse embryonic cortical and spinal cord neurons showed that halothane and eosin, a pump inhibitor, prolonged repolarization associated with spontaneous bursts of depolarization. These agents also prolonged the repolarization phases of electrically induced action potentials and of capsaicin-mediated Ca(2+)-dependent depolarization in mouse adult dorsal root ganglion neurons. In keeping with these findings, confocal microfluorimetry showed that halothane delayed clearance of intracellular Ca2+ accumulated by N-methyl-D-aspartate stimulation of single neurons.

Intrathecal ketorolac tromethamine produces analgesia after chronic constriction injury of sciatic nerve in rat.

PURPOSE: The study compared analgesic efficacy of intrathecally administered ketorolac tromethamine (K) and morphine hydrochloride (M) (in equimolar doses) in the chronic neuropathic pain model, induced by chronic constriction injury (CCI) of the sciatic nerve in rat. METHODS: Male Sprague-Dawley rats (n = 30) were anaesthetized with halothane and an intrathecal catheter was inserted to the mid-lumbar level of the spinal cord. On the 5th post-operative day, rats were anaesthetized with halothane and four ligatures were loosely applied around the right sciatic nerve. Seven days later, those animals were randomly divided into three groups and were injected with either saline, M (20 nmoles) or K (20 nmoles). Two pain responses (foot-withdrawal delay and hind paw elevation time) were measured on both sides using the radiant heat method. Further, thermal ("cold") allodynia was assessed by measuring of the total time of hind paw elevation in animals placed on the cold metal plate. RESULTS: Twenty nmoles of M and K injected intrathecally produced decrease of differential pain score calculated for both measured responses (hind paw withdrawal and hind paw elevation), compared with saline injected animals (P < 0.05). The reduction in pain response produced by K was less (P < 0.05). than the reduction in pain response observed in the animals receiving intrathecal M. Measurement of cold allodynia revealed that the animals in M and K injected groups demonstrated decreases in the total hind paw elevation time, when compared with saline-injected animals (P < 0.05). CONCLUSION: M and K produced hypoalgesia after intrathecal administration in rats with CCI, with M being more potent than K at an equimolar dose range. The analgesic effect of K was equal to equimolar doses of M for alleviation of cold allodynia.

Early effects of total hepatectomy on haemodynamic state and organ uptake of catecholamines in the pig.

In a previous study, we showed that plasma concentrations of catecholamines were increased during the anhepatic phase in pigs. In this study, we investigated if a constant depth of anaesthesia would prevent these changes and, if not, if the changes were caused by impaired extraction of catecholamines. We measured arterial and venous pressures, heart rate and cardiac output in 10 anaesthetized pigs. Hepatic arterial and portal venous flows were measured. Blood for measurement of catecholamines was sampled from carotid and pulmonary arteries and portal, hepatic and renal veins. After a 2-h observation period, the liver was removed and the circulation reconstituted. Measurements were made and samples obtained for another 2 h. Catecholamine concentrations increased 2-10-fold after hepatectomy. Before hepatectomy, noradrenaline was extracted by the lung (mean extraction ratio 23 (SEM 8)%) and the liver (30 (11)%); after hepatectomy, there was extraction by the kidney (24 (12)%) but extraction by the lung (29 (8)%) was unchanged. Before hepatectomy, adrenaline was extracted predominantly by the kidney (73 (5)%) and the liver (70 (6)%), with minimal extraction by the lung; after hepatectomy, extraction by the lung increased (25 (4)%) and decreased slightly in the kidney (56 (6)%). While mean arterial pressure did not change, heart rate increased by approximately 50% and cardiac index declined (ns) within 2 h after hepatectomy. There was a sharp increase in pulmonary vascular resistance after removal of the liver and changes correlated with increases in arterial plasma concentrations of catecholamines.

Persistent pain associated with long-term intrathecal morphine administration.

A 32-year-old man with chronic intractable right lower extremity pain unresponsive to multiple neurosurgical and pharmacologic treatments, including intrathecal morphine administration, was successfully treated with sciatic nerve block, discontinuance of opioid therapy, and psychologic interventions. Plasma and urine ratios of morphine metabolites morphine-3-glucuronide and morphine-6-glucuronide were analyzed at the beginning of our interventions, and the results indicated that morphine-3-glucuronide levels were significantly higher than morphine-6-glucuronide levels. The possible association between the observed morphine metabolite ratio and the intractable pain in patients resistant to opioids may have potential clinical implications.