Story 1 It wasn’t two months into my fellowship in Clinical Chemistry, working late after the routine staff had left for the day. The phone rings (BRNNG! - [old phone-1978]), and I answer. I recognize the voice on the line as a world famous endocrinologist; I had attended his lecture this week, so the voice was familiar. Doc asks “So, how’s the thyroxine reading machine working today?” (me: Pause - What??? Doesn’t this eminent physician know that there isn’t “a thyroxine reading machine”? There are extraordinarily talented people working to add the right chemicals, isotopes, separating the phases, and putting the correct fraction in the isotope counter, then using log-logit transformation to calculate the thyroxine concentration, then entering the result on a piece of paper to be attached to the patients chart.) I think some more…what is this very smart, eloquent, courteous clinician really asking?…AHA, he is asking me if we had any problems in the Lab today. Moyer learning experience #1, learn how to understand the question to get to the useful answer. He want’s to know if the result is correct. I ask if I can call him back in 3 minutes after I review everything (no more than three minutes because I know he will move on to another chart and not take my return call). So, less than three minutes later I call back to say I reviewed all the the quality checks, and all indicators suggest the result is correct. His response, another learning experience for Moyer, “OK, just wanted to check before I change my diagnosis. The result didn’t fit with my expectation, so now that I believe it to be real, I will reconsider my initial diagnosis. Thanks for your help.” Moyer to self: Thank you Doc X for that valuable learning experience.
Story2 - Laetrile for cancer treatment - one of the world's greatest hoax.
In the early 1950s, amygdalin and a chemical derivative named laetrile began to be promoted as alternative cancer treatments. While amygdalin and laetrile were never approved for use in the United States, it's promotion was highly touted by a fake literature; treatment with laetrile was readily available in Mexico. Promotion of laetrile to treat cancer has been described in the medical literature as a canonical example of quackery and as the slickest, most sophisticated, and the most remunerative cancer promotion in medical history. Numerous scientific studies found these compounds to not only be clinically ineffective in treating cancer, but also potentially toxic or lethal when taken by mouth due to cyanide poisoning.
Amygdalin is a naturally occurring chemical found in many plants, most notably in the seeds of apricots, bitter almonds, apples, peaches, cherries and plums, and in the roots of manioc. Amygdalin is a cyanogenic glycoside; each molecule includes a nitrile group, which can be released as toxic cyanide by the action of a beta-glucosidase. Ingestions of amygdalin will result in release of cyanide into the human body that may lead to cyanide poisoning.
Because many cancer patients came to our medical center insisting on being treated with laetrile, we decided to perform a clinical study with sufficient safeguards to ensure participating patients were protected from anticipated cyanide toxicity. Prior to implementation, the study plan underwent extensive Human Study review by both Mayo Clinic and the National Cancer Institute to ensure patient safety and propriety; the study was carried out under standard Phase I pharmaceutical research protocol. Six patients with advanced cancer were treated with laetrile at standard doses employed by laetrile advocates. Biological specimens were collected to facilitate a thorough evaluation of cyanic exposure. During and following completion of the study, none of the patients exhibited signs or symptoms of cyanide intoxication, all of the biological samples collected demonstrated elevated cyanide levels. The study concluded that exposure to laetrile predisposed patients to significant cyanide body burden that could dispose them to cyanide toxicity.
Reports from this study (A Pharmacologic and Toxicologic Study of Amygdalin. CG Moertel, MM Ames, JS Kovach, TP Moyer, JR Rubin, JH Tinker. J American Medical Association 1981;245:591594. and Pharmacology f Amygdalin (Laetrile) in Cancer patients. MM Ames, TP Moyer, JS Kovach, CG Moertel, Rubin. Cancer Chemother Pharmacol 1981;6:51-77.) and numerous others (see Wikipedia) clearly showed the danger associated with laetrile therapy resulting in it subsequent dismissal as a legitimate therapy.
Story 3 - A case of gout treatment causing regional enteritis.
A 33 year old woman presented with a history of gut pain, commonly known as regional enteritis. She recited a prior history of joint pain that was ultimately associated with excess ingestion of red meat. Her home town physician diagnosed these symptoms as gout, advised her to reduce her meat consumption, and prescribed the common anti-gout medication, Zyloprim. After several months her joint inflammation resolved, but then she started to experience enteritis. Over time she described the painful experience of kidney stones, which she collected and presented to her physician at the time of examination. These kidney stones were sent to the lab for analysis. Surprisingly, the stones she submitted were not comprised of the common constituents of kidney stones, which are typically uric acid, oxalic acid, or calcium oxalate.
This lab report raised interest among the clinicians attending this patient, which lead to a case conference where alternative possibilities were discussed. Her astute physician asked the question "could these be stones result from the administration of Zyloprim?" Zyloprim, generic name allopurinol, is an FDA approved drug similar in chemical structure to uric acid. Under normal circumstances allopurinol is well tolerated by patients and does not cause kidney stones. The lab performed additional chemical analysis on the submitted stones to prove that the chemical present in these stones was oxypurinol, a natural metabolite of Zyloprim that is normally readily eliminated in the urine. Reduction in Zyloprim dose was recommended, and over months, the incidence of Zyloprim-related nephrolithiasis diminished.
This was the first reported case of Zyloprim-related nephrolithiasis. RM Stote, LH Smith, JW Dubb, TP Moyer F Alexander, JLA Roth. Oxypurinol Nephrolithiasis In Regional Enteritis Secondary to Allopurinol Therapy. Annals of Internal Medicine. 1980;92:384-385.
Story 4 - Cyclosporin blood levels.
Cyclosporin, also spelled ciclosporin and cyclosporine, is a calcineurin inhibitor used as an immunosuppressant medication. It is taken orally or intravenously to reduce inflammation due to rheumatoid arthritis, psoriasis, Crohn's disease, nephrotic syndrome, eczema, and in organ transplant to prevent graft rejection. It is also used as eye drops for keratoconjunctivitis sicca (dry eyes).
Cyclosporin was isolated in 1971 from the fungus Tolypocladium inflatum and came into medical use in 1983. It is on the World Health Organization's List of Essential Medicines. In 2022, it was the 185th most commonly prescribed medication in the United States, with more than 2 million prescriptions.
Cyclosporin works by decreasing the function of lymphocytes. It does this by forming a complex to block the phosphatase activity of calcineurin, which in turn decreases the production of inflammatory cytokines by T-lymphocytes. Cyclosporin is used to treat and prevent graft-versus-host disease in bone marrow transplantation and to prevent rejection of kidney, heart, and liver transplants.
Cyclosporin was first used to interfere with graft vs host reaction following solid organ transplant by Dr. Thomas Starzl at the University of Pittsburg. Starzl's 1992 memoir explains that Cyclosporin was an epoch-making drug for solid organ transplantation. It greatly expanded the clinical applicability of such transplantation by substantially advancing anti-rejection pharmacotherapy. Put simply, the biggest limits of applying solid organ transplantation more widely is not the cost or surgical skill (as formidable as those are), but rather, the problem of graft rejection and the scarcity of donor organs. Cyclosporin was a major advancement against the rejection component of the challenge.
Within the context of the major anti-rejection advantages offered by Cyclosporin treatment, patient management while taking Cyclosporin is complicated by the widely variable metabolism of the drug. Cyclosporin metabolism is controlled by the genetically variable enzyme Cytochrome P4503A4. There is no standard dose that results in optimal therapy; there is wide intra-individual variability in metabolism. The enzyme also experiences variable inhibition by a wide variety of common dietary ingredients. For example, ingestion of grapefruit juice will interrupt metabolism that can result in Cyclosporin toxicity.
The solution to this problem was in the Lab. Shortly after introduction of Cyclosporin, blood level monitoring became the standard of practice. Dose adjustments based on blood concentration of Cyclosporin immediately after transplant became mandated. If a patient showed any signs of rejection or Cyclosporin toxicity, blood specimens were drawn immediately to determine blood concentration that defined whether dose adjustments were needed. With careful Cyclosporin blood level monitoring, during the period of the 1980's the rate of success of solid organ transplant rose from less that 50% to greater than 95%. Monitoring Cyclosporin blood concentration played a major role in this success.
Mayo Clinic Lab played a significant role in this medical advancement: (Moyer TP, Charlson JR, Ebnet LE. Improved chromatography of cyclosporine. Ther Drug Monit. 1986; 8(4):466-8. PMID:3824434. Moyer TP, Johnson P, Faynor SM, Sterioff S. Cyclosporine: a review of drug monitoring problems and presentation of a simple, accurate liquid chromatographic procedure that solves these problems. Clin Biochem. 1986 Apr; 19(2):83-9. PMID:3518993. Post GR, Moyer TP, Anderson CF, Sterioff S. Long-term cyclosporine for renal allograft patients does not cause nephrotoxicity. Transplant Proc. 1987 Feb; 19(1 Pt 2):1761-3. PMID:3079036. de Groen PC, McCallum DK, Moyer TP, Wiesner RH. Pharmacokinetics of cyclosporine in patients with primary biliary cirrhosis. Transplant Proc. 1988 Apr; 20(2 Suppl 2):509-11. PMID:3363652. Moyer TP, et al. Consensus Document: Hawk's Cay meeting on therapeutic drug monitoring of cyclosporine. Transplantation Proceedings. 1990; 22(3):1357-61. PMID:2190399.)
Story 5 - Hemochromatosis
British spelling Haemochromatosis, also know as iron overload disease, is unusual increased accumulation of iron in the body. This condition leads to organ damage involving the liver, pancreas, skin, heart, and joints. There are two predominant forms, hereditary hemochromatosis (i.e. familial) and transfusion-related iron overload. Patients with hemochromatosis may present with liver cirrhosis, hypogonadism, diabetes, meloderma (skin bronzing) or arthritis. Diagnosis require laboratory evaluation of blood and tissue samples
Primary hemochromatosis involves a genetic variation in the C282Y/C282Y loci of the hemochromatosis gene (HFE gene), an autosomal recessive gene on Chromosome 6. Preliminary diagnosis of iron overload utilizes the tests for serum transferrin saturation which measures the amount of iron bound to protein and serum ferritin which depicts the amount iron stored in the liver. Elevated values of both tests are typical for iron overload, but are not diagnostic for hemochromatosis. Follow up testing involves HFE gene evaluation. If liver damage is suspect, a liver biopsy may be collected to evaluate degree of liver damage, cirrhosis, or scarring. During liver biopsy evaluation, qualification of tissue iron content presented per gram of dry weight tissue correlated with age, known as the hepatic iron index, is diagnostic. (Ludwig 1993)
The solution to this diagnosis was in the Lab. The diagnosis of familial hemochromatosis requires demonstration of a gene abnormality at the C282Y/C282Y gene loci and an abnormal hepatic iron index. In a patient with past history of multiple blood transfusion, a normal gene finding but an abnormal hepatic iron index indicates transfusion-related iron overload.
References: Ludwig J, Batts KP, Moyer TP, Poterucha JJ. Advances in liver biopsy diagnosis. Mayo Clin Proc. 1994 Jul; 69(7):677-8. PMID:8015333. Ludwig J, Hashimoto E, Porayko MK, Moyer TP, Baldus WP. Hemosiderosis in cirrhosis: a study of 447 native livers. Gastroenterology. 1997 Mar; 112(3):882-8. PMID:9041250.
Story 6 - Tobacco Addiction Evaluation:
Tobacco use is one of the leading causes of preventable disease, disability, and death in the United States. Nicotine, present in tobacco products such as cigarettes, pipes, cigars, or chew, is an addictive substance that causes individuals to continue use of tobacco despite concerted efforts to quit.
Tobacco users engaged in programs to abstain from tobacco require support in the form of counseling, pharmacotherapy, and continuous encouragement. Occasionally, counselors may elect to monitor abstinence. The solution to this uncertainty is in the Lab by measurement of nicotine and metabolites in urine to verify abstinence. If results of biologic testing indicate the patient is actively using a tobacco product during therapy, additional counseling or intervention may be appropriate.
During treatment for nicotine addiction, quantification of urine nicotine and metabolites, while a patient is actively using a tobacco product, is useful to define the concentrations that a patient achieves through self-administration of tobacco. Nicotine replacement dose can then be tailored to achieve the same concentrations early in treatment to assure adequate nicotine replacement so the patient may avoid the strong craving they may experience early in the withdrawal phase. This can be confirmed by measurement of urine nicotine and metabolite concentrations at a steady state (2-3 days after replacement therapy is started). Once the patient is stabilized on the dose necessary to achieve complete replacement and responding well to therapy, the replacement dose can be slowly tapered to achieve complete withdrawal.
Nicotine is rapidly metabolized in the liver to cotinine, exhibiting an elimination half-life of 2 hours; it is usually detectable for 1-3 days after use. Cotinine exhibits an apparent elimination half-life of 15 hours; cotinine may be detected in urine for up to ten days after last use. Cotinine accumulates in urine in proportion to dose and hepatic metabolism (which is genetically determined); tobacco users excrete cotinine. Detection of nicotine and cotinine in urine indicates the the patient is using tobacco or is receiving high-dose nicotine patch therapy.
In addition to nicotine, tobacco products contain other alkaloids that can serve as unique markers of tobacco use. Two such markers are anabasine and nornicotine. Anabasine is present in tobacco products but not in nicotine replacement therapies. Nornicotine is present as an alkaloid in tobacco products and as a metabolite of nicotine. The presence of anabasine or nornicotine in urine indicates current tobacco use, irrespective of whether the subject is on nicotine replacement therapy. The presence of nornicotine without anabasine is consistent with use of nicotine replacement products.
Passive exposure to tobacco smoke can cause accumulation of nicotine metabolites in nontobacco users. Cotinine accumulates in urine L from passive exposure. Neither anabasine nor nornicotine accumulates from passive exposure.
Similar testing of blood can also be used for patient evaluation - see < https://www.mayocliniclabs.com/test-catalog/overview/82509#Clinical-and-Interpretive > for more information.
References: Dale LC, Hurt RD, Hays JT. Drug therapy to aid in smoking cessation. Tips on maximizing patients' chances for success. Postgrad Med. 1998;104(6):75-78, 83-84. Moyer TP, Charlson JR, Enger RJ, Dale LC, Ebbert JO, Schroeder DR, Hurt RD. Simultaneous analysis of nicotine, nicotine metabolites, and tobacco alkaloids in serum or urine by tandem mass spectrometry, with clinically relevant metabolic profiles. Clin Chem. 2002 Sep; 48(9):1460-71. Ebbert JO, Dale LC, Patten CA, Croghan IT, Schroeder DR, Moyer TP, Hurt RD. Effect of high-dose nicotine patch therapy on tobacco withdrawal symptoms among smokeless tobacco users. Nicotine Tob Res. 2007 Jan; 9(1):43-52.
Story 7 - Warfarin Genotyping
Warfarin (Coumadin) is an anticoagulant that has been the standard of care for more than 50 years to prevent deep vein thrombosis, pulmonary embolism, and treat thromboemboli. Millions of Americans initiate warfarin therapy annually, and currently there are few oral alternatives (Eliquis, Pradaxa, and Xarelto) available in the U.S. Since introduction, drugs such as Eliquis have become the preferred therapy for atrial fibrillation. However, because of the overriding need for effective anticoagulation, warfarin remains a commonly used drug in spite of the significant morbidity and mortality associated with its use. It is the second leading drug-related reason for emergency department visits and the most often cited reason for drug-related mortality.
Health care professionals monitor patients on anti-coagulation therapy using the INR (International Normalized Ratio) test. Based on the INR result, a healthcare professional decides if any change to warfarin dose is required. INR is a derivative of prothrombin time, a measurement of blood coagulation in the circulatory system used to determine the clotting rate of blood, which can be affected by anticoagulant usage, liver damage and Vitamin K levels. The preferred range of INR levels for a patient on anticoagulation therapy is usually between 2 and 3.
Reaching a stable, therapeutically effective dose of warfarin is difficult because it is dependent on multiple factors, including age, weight, diet, concurrent medications, and genetic variability in drug response. Most drug-related adverse events are due to problems in establishing the effective dose (i.e achieving a normal INR); it can take many weeks to evaluate, adjust, and stabilize the dose for an individual patient. During the dose adjustment phase, patients are at serious risk for hemorrhage or thrombosis. Event rates for bleeding or thromboembolism range as high as 16.5% and 25% during the first 6 months of warfarin treatment in usual care settings. The solution to this problem is in the Lab.
Over the past decade, variations in 2 genes have been shown to predict individual response to warfarin dosing. One gene determines the activity of the hepatic isoenzyme cytochrome P450 2C9 (CYP2C9), which plays a significant role in metabolizing S-warfarin into its inactive form. The other gene, VKORC1 (Vitamin K Epoxide Reductase subunit 1), determines the activity of the enzyme that produces the active form of vitamin K necessary for blood clotting. The CYP2C9 and VKORC1 polymorphisms account for more than one-third of the variance associated with stable therapeutic dosing. Simultaneous evaluation of a patient’s deoxyribonucleic acid (DNA) for allelic variations in these 2 genes is known as warfarin sensitivity genotyping.
A significant correlation has been demonstrated comparing steady-state warfarin dose and allelic variants of CYP2C9 and VKORC1 has been demonstrated by many previous reports. Although we were unable to show a statistically significant relationship between allelic variants and initial warfarin dose or dose escalation, an association was demonstrated between allelic variant and steady-state warfarin dose. White people show considerable variance in CYP2C9 allele types, whereas those of Asian and African descent infrequently carry CYP2C9 allelic variants. The VKORC1AA allele associated with high warfarin sensitivity predominates in those of Asian descent, whereas white people and people of African descent show diversity, carrying either the VKORC1BB allele associated with low warfarin sensitivity or the VKORC1AB or VKORC1AA alleles associated with moderate or high warfarin sensitivity, respectively. People of African descent predominantly carry the VKORC1BB allele associated with de-
creased warfarin sensitivity. 86% of those of Asian descent and 16% of white people carry the 2C9*1*1 VKORC1AA variant, predisposing them to wide swings in INR throughout drug initiation. An additional 9% of white people and a lesser percentage of those of
African descent carry the combination of CYP2C9 and VKORC1 allelic types associated with high sensitivity to warfarin. The incidence of the VKORC1 allele type in those of African descent suggests that 76% carry allelic variants associated with warfarin sensitivity requiring a higher than typical dose to maintain therapeutic INR.
Warfarin dosing guided by warfarin sensitivity genotyping improves patient outcome, predisposing them to lower risk adverse events while achieving steady state does more quickly.
References: Moyer TP, O'Kane DJ, Baudhuin LM, Wiley CL, Fortini A, Fisher PK, Dupras DM, Chaudhry R, Thapa P, Zinsmeister AR, Heit JA. Warfarin sensitivity genotyping: a review of the literature and summary of patient experience. Mayo Clin Proc. 2009 Dec; 84(12):1079. Epstein RS, Moyer TP, Aubert RE, O Kane DJ, Xia F, Verbrugge RR, Gage BF, Teagarden JR. Warfarin genotyping reduces hospitalization rates results from the MM-WES (Medco-Mayo Warfarin Effectiveness study). J Am Coll Cardiol. 2010 Jun 22; 55(25):2804-12.
