Guest Blogger: Cory Villanueva, Pharm.D. Candidate 2013, under the mentorship of Dr. Jeffrey Fudin. . Guest posts by Resident and Intern Pharmacists are reviewed, edited, and approved for accuracy by Dr. Jeffrey Fudin prior to posting.
Image: “Morphine – Endo Family Portrait”. 4andsic. February 24, 2011.
Available at: http://www.pharmer.org/images/domestic/endo-family
Introduction
Opioid serum levels can be very helpful in guiding treatment for patients with chronic pain, but there are several questions to consider when interpreting these levels. This discussion addresses firstly the question “What is the difference between serum free and serum total morphine?” We then define the correlation between morphine dose and serum free morphine level.
Serum free morphine may be ordered outside a clinic’s facility to its contracted labs. However, to be confident in the levels we obtain, the second question we assess is “How is a serum free morphine level obtained?” We will explain what lab procedure is used for serum free morphine and what steps are involved in obtaining serum free morphine.
The last question we address is “Can metabolism of morphine differ amongst patients?” Herein, we review recent research on the influence of genetic polymorphisms of specific enzymes upon the metabolism of morphine.
I. Serum “Free Morphine” versus “Total Morphine”
Morphine is metabolized primarily into two metabolites: major metabolite morphine-3-glucuronide (M3G) and minor metabolite morphine-6-glucuronide (M6G) (see Section III for further explanation of the metabolism of morphine). Essentially, “total morphine” is the sum of “free morphine” and morphine released after hydrolysis of M3G and M6G. “Total morphine” technically includes several additional metabolites which are present in very small quantities after metabolism: morphine-2,6-diglucuronide, morphine-3-etheral sulfate, normorphine, and normorphine-6-glucuronide. Indeed, because of neglecting these metabolites, we can understand that “total morphine” is already somewhat incomplete.
The percentage of morphine that is metabolized into M3G and M6G can vary within approximate ranges and the extent of glucuronidation that forms these metabolites can differ amongst patients. Further, using hydrolysis of M3G and M6G to then calculate total morphine adds additional uncertainty to the analysis of total morphine.
More recently, specific measurements of M3G, M6G, and free morphine are used to determine total morphine. Total morphine is primarily relevant in analysis of free morphine: total morphine ratios for post-mortem cases. These cases may involve heroin morphine, or opium overdoses, codeine or the determination of whether or not morphine was the cause of death. M3G concentrations have been shown to exceed free morphine within two hours after a dose is taken; accordingly, if free morphine constitutes most of the total morphine in a patient’s plasma (high free morphine: total morphine ratio), then it is more likely that the death was acute and the patient did not have time to metabolize the medication.
Free serum morphine for a 24-hour total dose of 40mg immediate release morphine should be 11.1 +/- 8.4 ng/mL.2 Free serum morphine for a 24-hour total dose of 100mg extended release morphine should be 36.9 +/- 15.5ng/mL.2
II. Obtaining a Serum Free Morphine Level
There are many published methods for determining morphine and its glucuronide metabolites3,4,5:
- gas chromatography (GC)
- high performance liquid chromotography (HPLC)
- gas chromatography / mass spectrometry (GC / MS)
- liquid chromatography / mass spectrometry (LC/MS)
- liquid chromatography coupled with tandem mass spectrometry (LC / MS / MS)
For decades morphine analysis included methods involving solvent extraction followed by thin-layer chromatography; this allows the detection of other abused opioids at the same time.6 The need for widespread screening for work-place and military encouraged the development of immunoassays for detecting urine opioids; commercial kits are based on radioimmunoassay (RIA), enzyme immunoassays (EIA), and fluorescence polarized immunoassays (FPIA).1
Enzyme-Linked Immunosorbent Assay (ELISA) can provide a preliminary analytical result, but a more specific chemical process such as GC/MS must be performed to confirm the result and to allow us to quantify the morphine level.6 Morphine is extracted from serum by solid-phase chromatography. Morphine , M3G, and M6G may be extracted from the same column, but because they differ in polarity different elution solvents may be necessary. Hydrolysis of M3G and M6G may be done with heat and acidic conditions or done with β-glucoronidase enzyme.
Mass spectrometric analysis is very sensitive to morphine and other opiates in biological fluids. High Performance Liquid Chromatography (HPLC) avoids problems of complexity that are encountered with preparing samples for GLC methods. Detection of morphine and its conjugates by UV or visible spectrophotometry may be vulnerable to interference; for low sensitivity levels, flurorescence and electrochemical detection is preferable. Some methods simultaneously determine morphine and M3G and M6G using fluorescence detection or a combination of fluorescence and electrochemical detectors in series.
III. The Influence of Polymorphisms upon Morphine Metabolism and Analgesia
Various enzymes are involved in morphine metabolism, transport, and penetration through the blood brain barrier (BBB). Genetic polymorphisms of these enzymes may account for the differences in morphine metabolism as well as analgesic response amongst different patients. We continue our discussion below in focusing upon specific steps in the metabolism of morphine which may be affected by these polymorphisms.
Glucuronidation by UGT Enzymes
The oral bioavailability of morphine ranges from 19-47% and is significantly affected by first pass metabolism by the liver.1 The main pathway by which morphine is metabolized in humans is glucuronidation by UDP-glucuronosyltransferase (UGT) enzymes. The human UGTs are divided into four families (UGT 1, 2, 3, and 8) according to homology amongst DNA sequences.7 The ability to glucuronidate morphine differs amongst individuals. UGT2B7 is the major isoform responsible for 3- and 6-glucuronidation of morphine and it is theorized that genetic polymorphisms of UGT2B7 are responsible for variations in the ability to glucuronidate.8 Such a polymorphism provides reason for increased glucuronidation in Chinese patients compared to Caucasians.1
UGT2B7 variants may alter drug metabolism and therefore disease risk.7 Researchers have found that a common UGT2B7 haplotype (made up of ten single-nucleotide polymorphisms) increases enzyme activity and may be the reason why drug metabolism may differ amongst UGT2B7 substrates.
Morphine Metabolites
UGT2B7 glucuronidates the 3-OH phenolic group to form major metabolite morphine-3-glucuronide (M3G) and glucoronidates the 6-OH alcoholic group to form minor metabolite morphine-6-glucuronide (M6G) at a ratio of 5:1 for M3G:M6G.7 Only small amounts of the following metabolites are produced: morphine-2,6-diglucuronide, morphine-3-etheral sulfate, normorphine, and normorphine-6-glucuronide. 44-55% of a morphine dose is converted to M3G, 9-10% to M6G, 4% excreted as normorphine and its glucuronide metabolites, 8-10% is excreted in urine unchanged, and the remainder is excreted through feces, perspiration or is formed into the other minor metabolites.7 M3G does not help with analgesia but may actually antagonize the effects of morphine and cause myoclonus / seizure as well as allodynia (pain due to a stimulus that usually does not cause pain).
Other genetic polymorphisms affect the metabolism of morphine, as well. Polymorphisms of the μ-opioid receptor gene (OPRM1) may affect the therapeutic efficacy of morphine and polymorphisms of Catechol-O-MethylTransferase (COMT) affect pain perception as COMT is responsible for the metabolism of dopamine, epinephrine, and norepinephrine.7
Compounds may be competitive substrates for UGT2B7 and thereby can reduce the formation of M3G and M6G. Studies have found that Oxazepam inhibits morphine conjugation, ranitidine may decrease M3G:M6G ratio, and tricyclic antidepressants have inhibited morphine UGT in human liver microsomal preparations.7
Penetrating the Blood Brain Barrier
After oral administration of morphine, the plasma AUC (Area Under the concentration-time Curve) ratio for M6G:morphine is 9:1 and for M3G:morphine is 50:1.7 Most analgesia is due to the M6G metabolite rather than from parent morphine. Studies in rats, however, have found that M6G is 7.5 times less permeable through the BBB relative to morphine.5 The theory that is particularly relevant to a discussion of genetic polymorphisms is that efflux transporter P-glycoprotein (ATP-binding cassette, subfamily B, member 1 (ABCB1)) pumps morphine out the central nervous system and has a significant influence on penetration of the BBB.7 Polymorphisms of ABCB1 have realized varied analgesic responses in cancer patients.
Conclusion
Serum free morphine levels are part of a larger picture in which medication dosing, lab procedure validity, and theory of drug metabolism are considered. Knowing how to interpret these levels can improve the safety and efficacy of the treatment we provide. Moreover, a working knowledge of therapeutics, the tolerance level of a patient antemortem are very important when making a determination of death postmortem.
All references appear below.
Cory Villanueva is a Student Pharmacist at the Albany College of
Pharmacy and Health Sciences. He currently works at CVS
Caremark retail pharmacy in Cohoes, NY part time as
a Student Intern. His academic interests are
managed care and psychiatric pharmacy.
References:
1. Andersen G, Christrup L, Sjøgren P. Relationships among morphine metabolism, pain and side effects during long-term treatment: an update. J Pain Symptom Manage. 2003 Jan;25(1):74-91. Available from: http://www.jpsmjournal.com/article/S0885-3924(02)00531-6/abstract
2. Fudin, Perkins. Opioid Pharmacokinetics and Expected Metabolites. Rev 04/2011. Available from: https://paindr.com/wp-content/uploads/2012/05/Opioid-Serum-Predictability-and-Metabolites.pdf
3. Klepstad, P., et al., Day-to-day variations during clinical drug monitoring of morphine, morphine-3-glucuronide and morphine-6-glucuronide serum concentrations in cancer patients. A prospective observational study. BMC Clin Pharmacol, 2004. 4: p. 7. Available from: http://www.biomedcentral.com/1472-6904/4/7
4. Al-Asmari, A.I. and R.A. Anderson, Method for quantification of opioids and their metabolites in autopsy blood by liquid chromatography-tandem mass spectrometry. J Anal Toxicol, 2007. 31(7): p. 394-408. Available from: http://jat.oxfordjournals.org/content/31/7/394.long
5. Bogusz, M.J., R.D. Maier, and S. Driessen, Morphine, morphine-3-glucuronide, morphine-6-glucuronide, and 6-monoacetylmorphine determined by means of atmospheric pressure chemical ionization-mass spectrometry-liquid chromatography in body fluids of heroin victims. J Anal Toxicol, 1997. 21(5): p. 346-55. Available from: http://jat.oxfordjournals.org/content/21/5/346.full.pdf
6. Hans Brandenberger, Robert A. A. Maes. Analytical toxicology: for clinical, forensic, and pharmaceutical chemists. New York: de Gruyter, 1997. Available from: http://books.google.com/books?id=ZhYtynyC4kAC&printsec=frontcover#v=onepage&q&f=false
7. De Gregori S et al. Morphine metabolism, transport and brain disposition. Metab Brain Dis. 2012 Mar;27(1):1-5. doi: 10.1007/s11011-011-9274-6. Epub 2011 Dec 24. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3276770/
8. Di YM, Chan E, Wei MQ et al (2009) Prediction of deleterious nonsynonymous single-nucleotide polymorphisms of human uridine diphosphate glucuronosyltransferase genes. AAPS J 11:469–480 (PDF available per request)
9. Holthe M, Klepstad P, Zahlsen K et al (2002) Morphine glucuronideto- morphine plasma ratios are unaffected by the UGT2B7 H268Y and UGT1A1 *28 polymorphisms in cancer patients on chronic morphine therapy. Eur J Clin Pharmacol 58:353–356 (PDF available per request)
WHAT DOES 220 NG/ML MEANS AND YOU TELL METHR TIME THEY TOOK THIS MUCH
Angel, I’m not sure what you are asking. Did you have a blood morphine level that came back at 220ng/mL?
I am charged with driving while intoxicated. I have been treated for cronic pain for many years. I am told that I had 41ng ml of morphine in my blood. Is this considered intoxicating.
41ng/mL equates to about 100mg of oral morphine per day in divided doses. If you take this medication regularly and have a prescription, and are tolerant to opioids, depending on the state, there is a weak case against you unless there were other drugs and/or alcohol involved.
Hello, I just received my friends post mortem toxicology report. He had an alcohol level of .249 and the morphine level was .85. Since he wasn’t prescribed any morphine drugs, I am guessing he purchased something like oxycontin from his ‘friends’. Could you tell me if this was a lethal combination (or could he have died of other factors). Also, the morphine level of .85. How would that translate to street pills like oxycontin? Thank you for your article. I think this is the only place I can get these answers as the coroner did not know what .85 meant.
This cannot be answered without the actual report.
Hi – Is it possible for you to tell me what .85 means? Thank you.
Could be mcg/ml, ng/mL, ng/L, etc. Could be congregated morphine or free morphine, and depends on where the blood came from (femoral artery, heart, vitreous fluid etc.). Asyou can see, there are too many variables to give you a quick simple answer.
My beloved mother in law, Marjorie, passed away at 1 pm today. She had been given multiple doses of morphine for pain in the ICU. Are levels routinely monitored in the hospital? What is the turn around time? Does the patient’s family have to insist that the levels be monitored? Can the genetic differences in metabolism be screened in advance if morphine is likely to be needed? She was incoherent and delirious-how can I be sure this was monitored by hospital staff appropriately?
Mary, Here are the answers to your questions:
Are levels routinely monitored in the hospital? Almost never
What is the turn around time? See above, but it would take about a week
Does the patient’s family have to insist that the levels be monitored? I’ve never seen this happen, nor is it the standard of care. Instead, there are other parameters monitored such as oxygen saturation respiration, and pulse
Can the genetic differences in metabolism be screened in advance if morphine is likely to be needed? Yes
She was incoherent and delirious-how can I be sure this was monitored by hospital staff appropriately? There would need to be an expert review of the medical record
my son died of acute morphine intoxication morphine was elevated at 163 what does this mean?
In a case where post mortem morphine blood levels
are 490 ng/ml free and 1600 ng/ml total, and the deceased had no opiate addiction (therefore no tolerance issues), are these levels considered toxic or lethal? Autopsy results show no issues with iiver and kidney function.
Thanks.
Zee, There are many factors to be considered to answer this question.
What does 47ng/ml of morphine mean in the blood
It generally means the patient is taking 120mg of morphine orally per day.
It was a great article although it would be nice if you could give the basics in layman’s terms so us patients with chronic pain and are on opioids might understand a little better of why and what the difference is between levels and tolerant for example if a patient is levels are high and the patient claims he is still in pain what does this mean when the doctor is trying to figure out the Dosefor the same patient. Thank you. Sincerely yours a jim
This is a quite technical, but very well-developed and important article. Well-done, Cory. Morphine has been in use for so long that most practitioners, and probably plenty of pharmacists, do not appeciate the complexities and individual patient differences regarding its metabolism and effects. More good reasons why setting dosing limits, especially those based on so-called morphine-equivalent dosing, does not make good sense — medically or pharmacologically.
Dr. Leavitt, thank you for the great feedback! It was certainly fascinating to review the literature on the complexities of morphine metabolism. I agree that it makes a timely argument against dosing limits that ought to help us advocate for patients with chronic pain.
Cory,
This is a nice review. In addition to the mechanisms you mention above there are genetic differences in Mu opioid receptors (mutation A118 G)that can also account for differences in the pain control derived from morphine. In one study, patients with this mutation at the Mu receptor required 3 times the morphine dose than those without the mutation to receive the same pain control. In addition, it appears that this mutation at the Mu receptor may be involved in promoting cancer growth and progression (animal studies).
There is likely other mechanisms that we are not aware of that also could play a role in how we as humans respond to morphine; but I guess that leaves further opportunities to investigate 🙂
Nicely done!
Dave
Dr. Craig,
Thank you for your feedback and your insight. Your thoughts on the genetic difference in the mu receptor are very interesting. These ideas may eventually support a model of personalized medicine in which we can better anticipate the patient’s responsiveness to morphine or propensity to adverse effects.