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INVITED REVIEW The Determination of Insulin Overdose in Postmortem Investigations Laura M. Labay, Casey P. Bitting, Kevin M. Legg, Barry K. Logan ABSTRACT The analysis of biological specimens for the presence of exogenous insulin is of special interest in select postmortem investigations. Insulin analogues are primarily used to mediate the regulation of blood glucose concentrations; however, their use has also been implicated or suspected as a cause of death in suicides, accidents, and homicides. Toxicological analysis for these compounds is challenging due to the large molecular weight, the limited stability of insulin in whole blood, and complexities associated with sample preparation and instrumental testing. As a consequence, determination of insulin in postmortem specimens is not routinely offered by most forensic toxicology ODERUDWRULHV)RUHQVLFGHDWKLQYHVWLJDWLRQLVIXUWKHUFRPSOLFDWHGE\LQWHUSUHWDWLYHGLI¿FXOWLHVVXFKDVWKHIUHTXHQWDEVHQFHRIDQDWRPLFDO ¿QGLQJVFRQFHQWUDWLRQLQWHUSUHWDWLRQLQNQRZQLQVXOLQXVHUVDQGDGGUHVVLQJWKHLPSDFWRIFKHPLFDOLQVWDELOLW\DQGSRVWPRUWHPUHGLVWULEXtion. There are ongoing efforts, however, to develop and validate robust methods that may be used for this analysis on these challenging VDPSOHVDQGWKDWDUHFDSDEOHRIZLWKVWDQGLQJVFLHQWL¿FDQGOHJDOVFUXWLQ\IRUIRUHQVLFXVH,QWKLVUHJDUGLQUHFHQW\HDUVPHWKRGVIRUWKH detection of exogenous insulin in postmortem samples have been reported and results of this testing has been published in a handful of cases. The purpose of this article is to review the primary functions of insulin, the disease states associated with the therapeutic use of exogenous insulin, the current state of laboratory testing, and to provide case summaries that summarize the timeline of advancements DQGXQGHUVFRUHWKHLPSRUWDQFHRIWKLVZRUNAcad Forensic Pathol. 2016 6(2): 174-183

AUTHORS

Laura M. Labay PhD F-ABFT, NMS Labs - Toxicology



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Casey P. Bitting DO, University of New Mexico School of Medicine - Pathology  

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Kevin M. Legg PhD, Center for Forensic Science Research and Education 

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Barry K. Logan PhD F-ABFT, NMS Labs - Toxicology 

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CORRESPONDENCE

Barry K. Logan MD, 3701 Welsh Rd, Willow Grove PA 19090, [email protected]

 FINANCIAL DISCLOSURE

7KHDXWKRUVKDYHLQGLFDWHGWKDWWKH\GRQRWKDYH¿QDQFLDOUHODWLRQVKLSVWRGLVFORVHWKDWDUHUHOHYDQWWRWKLV manuscript

DISCLOSURES

7KHDXWKRUVUHYLHZHUVHGLWRUVDQGSXEOLFDWLRQVWDIIGRQRWUHSRUWDQ\UHOHYDQWFRQÀLFWVRILQWHUHVW

FUNDING

None

KEYWORDS

Forensic pathology, Insulin analogues, C-peptide, Toxicology, Overdose, Analytical testing, Cause and manner of death, Vitreous humor, Postmortem, Suicide, Homicide, Mass spectrometry, Liquid chromatography, Immunoassay

INFORMATION

ACADEMIC FORENSIC PATHOLOGY: THE OFFICIAL PUBLICATION OF THE NATIONAL ASSOCIATION OF MEDICAL EXAMINERS (ISSN: 1925-3621) ©2016 Copyright Academic Forensic Pathology Incorporated Submitted for consideration on 19 Apr 2016. Accepted for publication on 24 May 2016

Page 174

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INVITED REVIEW INTRODUCTION

using alternative biological matrices, most notably YLWUHRXV ÁXLG   ,W LV OLNHO\ WKDW WKHVH DQDO\WLFDO advancements, in conjunction with the knowledge base that already exists for insulin biochemistry and physiology, will allow for more complete forensic investigations of suspected insulin-related deaths. This review article will summarize advances in technology that support the investigation of forensic cases where insulin use is suspected to have contributed to death.

The investigation of death due to exogenous insulin administration is complicated by the lack of tests that have been validated on autopsy specimens. This is due to a combination of factors including cost, pre-analytical issues regarding specimen handling, and pronounced analytical challenges. Considering insulin toxicity as a cause of death and then accurately determining manner of death requires a systematic investigation often coupled with a history of insulin abuse DQGRUPLVXVHDQGFKHPLFDOFRQÀUPDWLRQRIWKHSUHVence of the exogenous compound. Considering these requirements, it stands to reason that a proportion of WKHVHGHDWKVOLNHO\JRXQUHFRJQL]HGRUPLVFODVVLÀHG From a toxicological perspective, a key contribution to the investigations would be the ability to determine whether exogenous insulin is present and if possible, WR GHWHUPLQH WKH FRQFHQWUDWLRQ DQG LWV VLJQLÀFDQFH The major obstacle to this has been that traditional methods such as immunoassays used in clinical settings are typically not amenable for postmortem specimens due to interferences with hemolyzed samples in immunoassay methods. In the last decade, improvements in sample preparation and instrumentation have made progress in the detection and differentiation of insulin analogues in both insulin preparations and medical equipment (intravenous lines, and bags) and S

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Insulin Structure and Functions Proinsulin, the precursor molecule of insulin, is a pepWLGH WKDW LV V\QWKHVL]HG LQ WKH ơ FHOOV RI WKH SDQFUHas. It is cleaved by the action of a series of proteolytic enzymes to form insulin. This step removes the DPLQRDFLGUHVLGXHWHUPHG&SHSWLGHEHWZHHQWKH DPLQRDQGK\GUR[\WHUPLQDOHQGV7KHÀQDOLQVXOLQ SHSWLGHFRQVLVWVRIDDPLQRDFLGƠFKDLQ FDUER[\ WHUPLQDOHQG DQGDDPLQRDFLGơFKDLQ WKHDPLQR terminal end) held together by two interchain disulÀGHERQGV7KHƠFKDLQFRQWDLQVDQDGGLWLRQDOVLQJOH LQWUDFKDLQGLVXOÀGHERQG Figure 1) (4). In response to rising blood glucose concentrations, insulin is excreted with C-peptide in equimolar quantities into the portal circulation (5). S

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Figure 1: Primary peptide structure of human insulin adapted from USNLo. EXUBERA - insulin human drug label information, 2008 (4).

Page 175 Labay et al. • Insulin in Postmortem Investigations ACADEMIC FORENSIC PATHOLOGY: THE OFFICIAL PUBLICATION OF THE NATIONAL ASSOCIATION OF MEDICAL EXAMINERS ©2016 Academic Forensic Pathology Incorporated

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INVITED REVIEW Insulin is a vital hormone that has numerous anabolic physiological functions including stimulating DNA and RNA synthesis, oxidative phosphorylation, intracellular transport, lipogenesis, and glucose utilization. Foremost in allowing these actions to occur, insulin allows glucose to enter cells where it can be metabolized and used to drive cellular activity. Insulin also promotes anabolic processes by inhibiting protein catabolism and decreasing the release of glucagon, a FDWDEROLF KRUPRQH IURP SDQFUHDWLF Ơ FHOOV ,Q FRQtrast to insulin, glucagon promotes the breakdown of glycogen, the storage form of glucose. The two hormones, working in opposition, serve to stabilize glucose blood concentrations so that they are maintained within appropriate limits (6-8). Insulin Therapy Problems with insulin production and utilization charDFWHUL]HW\SHVDQGGLDEHWHVPHOOLWXVUHVSHFWLYHO\ 7\SH  '0 7'0  LV FDXVHG E\ DXWRLPPXQH GHVWUXFWLRQRIWKHLQVXOLQSURGXFLQJSDQFUHDWLFơFHOOV resulting in low or absent blood insulin concentrations, and presents in childhood or early adulthood. 7\SH'0 7'0 W\SLFDOO\SUHVHQWVODWHULQOLIHDQG is characterized by insulin resistance in peripheral tissues (i.e., muscle, fat, and liver), diminishing insulin excretion and increased hepatic glucose production. 7UHDWPHQWIRU7'0DQGIRUDGYDQFHG7'0LVLQVXlin injection (9). :KLOH WKH V\PSWRPV RI7'0 KDYH EHHQ GHVFULEHG IRU FHQWXULHV WKH SDQFUHDWLF KLVWRORJLFDO ÀQGLQJV RI 7'0 ZHUH ÀUVW GHVFULEHG LQ  E\ WKH *HUPDQ SDWKRORJLVW0DUWLQ6FKPLGWLQDQDXWRSV\VSHFLPHQ RID\HDUROGFKLOGZLWKGLDEHWHV  7KH1REHO 3UL]HZDVODWHUDZDUGHGWR%DQWLQJDQG%HVWZKRÀUVW

LVRODWHGLQVXOLQIURPWKHSDQFUHDVLQDOORZLQJ IRUSXULÀFDWLRQRIDQLPDOLQVXOLQIRUWUHDWPHQW   While this therapy was life-saving, the administration of animal insulin is associated with the development RIDQWLERGLHVWRWKHH[RJHQRXVLQVXOLQPROHFXOH   6LQFHWKHQWKHGHYHORSPHQWDQGXVHRIUHFRPELQDQW human insulin has resulted in a dramatic decrease in V\PSWRPDWLFLPPXQHUHVSRQVHVWRLQVXOLQ   /LNHSXULÀHGDQLPDOLQVXOLQUHFRPELQDQWKXPDQLQsulin, or insulin analogues, are exogenous types of insulin because these are made outside of the human body. Insulin analogues are available in several forms with molecular structures similar to endogenous human insulin, some differing by as little as one amino DFLG6\QWKHWLFKXPDQLQVXOLQ +XPXOLQ LVSURGXFHG in bacteria or yeast and is structurally identical to endogenous human insulin. Exogenous insulins are clasVLÀHGDVUDSLGDFWLQJLQWHUPHGLDWHDFWLQJRUORQJDFWing depending on the speed of onset and duration of activity (Table 1). Chemical and pharmacological properties of the insulins are detailed in Table 2. Specimen Handling In a postmortem assessment, the most common matrix collected and then submitted for toxicological evaluation is blood. Unfortunately, due to the variations associated with postmortem blood such as putrefaction, hemolysis, and clotting, this specimen type is not amenable to insulin analysis by immunoassay platforms. The use of serum or plasma samples that have been saved by the hospital, especially if drawn within minutes or hours of the suspected time of insulin intoxication, and if refrigerated or frozen, may be viable IRU LPPXQRDVVD\ WHVWLQJ 6WXGLHV VKRZ WKDW  WR RILQVXOLQLVUHFRYHUDEOHIURPVHUXPRUSODVPD

Table 1:,QVXOLQ&ODVVL¿FDWLRQE\2QVHW3HDNDQG'XUDWLRQ &ODVVLÀFDWLRQ

Onset

Peak

Duration

Rapid-Acting

PLQ

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KRXUV

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PLQ

2-5 hours

8SWRKRXUV

Intermediate-Acting

PLQKRXUV

KRXUV

Up to 24 hours

Long-Acting

45 min-4 hours

0LQLPDO

Up to 24 hours

Page 176 Labay et al. • Insulin in Postmortem Investigations ACADEMIC FORENSIC PATHOLOGY: THE OFFICIAL PUBLICATION OF THE NATIONAL ASSOCIATION OF MEDICAL EXAMINERS ©2016 Academic Forensic Pathology Incorporated

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INVITED REVIEW DIWHUVL[GD\VRIUHIULJHUDWLRQDWƒ&+RZHYHULQVXOLQLVQHDUO\FRPSOHWHO\GHJUDGHGE\IRXUWRÀYHGD\V DWURRPWHPSHUDWXUH  7KXVDQ\ELRORJLFDOVSHFimen collected for insulin analogue testing should be collected in as timely a manner as possible relative to the initiation of the investigation and immediately VWRUHG IUR]HQ QR ZDUPHU WKDQ ƒ& DQG LGHDOO\ DW ƒ&  Analytical Methods 6HYHUDOPHWKRGVFDSDEOHRIGHWHFWLQJDQGTXDQWLI\LQJ insulin have been reported. The insulin radioimmunoassay (RIA) was published by Berson and Yalow in   7KHVHQVLWLYLW\DQGUHSURGXFLELOLW\RI5,$ soon established it as the method of choice for most insulin investigations. The technique may also be employed for C-peptide determinations. Disadvantages to RIA, however, is that the test is time-consuming, subject to interferences, and requires laboratory personal to work with radiolabeled agents. Today, commercially available enzyme-linked immunosorbent DVVD\ (/,6$ NLWVKDYHEHHQGHVLJQHGDQGGHYHORSHG to quantitatively measure human insulin and C-peptide in serum and/or plasma samples. The advent of these assays has improved sensitivity and reproducibility and, from a laboratory perspective, has lowered cost and reduced turnaround time. From a forensic perspective however, these tests may QRWVXIÀFHHVSHFLDOO\LQWKHDEVHQFHRIVXSSRUWLYHDQamnestic case information. This is principally because current ligand-binding tests are unable to deliver discriminatory quantitative results from the laboratory to

the death investigator. Immunoassay cross-reactivity with nontarget compounds, dynamic range limitations and, most importantly, the ability to unambiguously differentiate endogenous insulin from recombinant pharmaceutical analogues remains likely unsolvable ZLWK WUDGLWLRQDO DQWLERG\EDVHG LPPXQRDVVD\V    %HFDXVH RI WKLV WKH LGHQWLÀFDWLRQ RI WKH LQVXlin analogue should be made by a technique such as liquid chromatography tandem mass spectrometry /&0606 WKDWSURYLGHVWKHDELOLW\WRGLVFULPLQDWH between the various synthetic analogues. Becoming increasingly popular for clinical endocrinology and VSRUWV PHGLFLQH WHVWLQJ /&0606 DVVD\V DUH QRW VXEMHFW FURVVUHDFWLYLW\ SURYLGH KLJKHU VSHFLÀFLW\ if not sensitivity, and are amenable to multiplexing PXOWLSOH WDUJHWV LQ D VLQJOH WHVW    )URP D UHSRUWLQJVWDQGSRLQWDZHOOGHVLJQHG/&0606DVVD\ should eliminate the concern that structurally similar compounds cross react, and therefore enable distinct LGHQWLÀFDWLRQDQGTXDQWLÀFDWLRQRIHLWKHUHQGRJHQRXV human insulin or the analogous recombinant varieties. 3URWHLQV FDQ EH VLPSO\ GHÀQHG DV ELRORJLFDO PROecules that perform cellular functions. Ultimately, protein function is dictated by amino acid sequence leading to highly specialized tertiary and/or quaternary structure. While all common pharmaceutical analogues have been developed with slight structural differences (Table 3) to achieve various desired pharmacokinetic and pharmacodynamic properties, the ultimate biological function of these insulin analogues LVWKHUHJXODWLRQRIEORRGJOXFRVHFRQFHQWUDWLRQV   +RZHYHUEHFDXVHRIWKHLUYHU\VLPLODUPROHFXlar characteristics, including mass and structure, these

Table 2: Chemical and Pharmacological Properties Trade Name

Empirical Formula

0ROHFXODU Weight (Da)

Onset of Action

Peak Action

Effective Duration

0D[LPXP Duration

Insulin (human)

+XPXOLQ

C+N65O66



KU

2-4 hr

6-8 hr

---

Lispro

+XPDORJ

C+N65O66



PLQ

PLQKU

KU

4-6 hr

Aspart

NovoLog

C256+N65O66

5825.8

PLQ

PLQKU

KU

4-6 hr

*OXOLVLQH

Apidra

C258+N64O66



PLQ

PLQKU

KU

4-6 hr

*ODUJLQH

Lantus

C+NO66



KU

KU

KU

KU

Detemir

Levemir

C+ON6466



6-8 hr

KU

KU

~2 hr

Compound Name

Page 177 Labay et al. • Insulin in Postmortem Investigations ACADEMIC FORENSIC PATHOLOGY: THE OFFICIAL PUBLICATION OF THE NATIONAL ASSOCIATION OF MEDICAL EXAMINERS ©2016 Academic Forensic Pathology Incorporated

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INVITED REVIEW compounds also have similar analytical characterisWLFVVSHFLÀFDOO\FKURPDWRJUDSKLFUHWHQWLRQWLPHDQG fragmentation spectra. Taken together, clear differentiation remains a challenge even for modern LC0606 EDVHG DSSURDFKHV UHTXLULQJ FDUHIXO PHWKRG optimization, thorough method validation, and strict quality control. ,Q UHFHQW \HDUV VHYHUDO /&0606 EDVHG PHWKRGV have been published. The most commonly employed WHFKQLTXH FRPELQHV ORZ ÁRZ FKURPDWRJUDSK\ PLcrobore or nanobore) with anti-insulin antibody imPXQRSXULÀFDWLRQ  :KLOHWKHVHDSSURDFKHV have seen some success, complex sample preparation VWHSV DVVRFLDWHG ZLWK LPPXQRFDSWXUH SXULÀFDWLRQ as well as lengthy run times, lack of robustness, and UHSURGXFLELOLW\ DVVRFLDWHG ZLWK ORZ ÁRZ VHSDUDWLRQV make these approaches largely unsuitable for forenVLF DQDO\VLV    0RUH UHFHQW DSSOLFDWLRQV KDYH RPLWWHG LPPXQRSXULÀFDWLRQ LQ OLHX RI PXOWLGLPHQVLRQDOFKURPDWRJUDSKLFSXULÀFDWLRQDVZHOODVLQWURGXFLQJ 06OHYHO VHSDUDWLRQ YLD LRQ PRELOLW\ PDVV VSHFWURPHWU\    +RZHYHU QR VLQJOH DSSURDFK has successfully integrated a straightforward, production-compatible, preparation method with unequivocal, unambiguous, discrimination between insulin and the recombinant analogues. Combining additional

DSSURDFKHVDQGDGYDQFHVVXFKDVLPPXQRSXULÀFDWLRQ automation, novel column chemistries and preparation techniques (e.g., enzyme digestion) are likely to provide a solution in the near future (28). Interpretation of Findings There are two main patient populations to consider: those that are not prescribed insulin and those that are prescribed insulin. In the former group, if exogenous LQVXOLQ LV LGHQWLÀHG ï UHJDUGOHVV RI FRQFHQWUDWLRQ ï this is substantive evidence that insulin may have at least contributed to death, especially in the absence of a more competent cause. In the latter population, it might be expected for the analytical method to detect an exogenous insulin. Quantitative analysis to determine concentration would be warranted in this circumstance and should be compared to concentrations corresponding to compliant therapeutic use when available for that particular specimen type. Another interpretive avenue is the consideration of the insulin to C-peptide (I:C) ratio. This ratio has been used to indicate the use or administration of exogeQRXVLQVXOLQ  ,QOLYLQJLQGLYLGXDOVWKHSODVPD KDOIOLIH RI LQVXOLQ LV EHWZHHQ ÀYH WR HLJKW PLQXWHV while the plasma half-life of C-peptide is between ten

Table 3: Amino Acid Sequence of Human and Insulin Analogues Compound

Chain

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,QVXOLQ +XPDQ

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