Published: Journal of Analytical Toxicology, ISSN 0146-4760, Volume 25, Number 5, July/August, pp. 356-357

Letter To The Editor:

Response to The Presence of Gamma-Hydroxybutyric Acid (GHB) in Postmortem Biological Fluids

To the Editor:
We were intrigued by the Letter to the Editor by Elliott (1) regarding the production of GHB in postmortem biological fluids and felt compelled to respond. The author reports much higher endogenous postmortem-production GHB concentrations than have been previously reported, especially in urine specimens. However, several important details of this study, such as the time between death and the collection and storage conditions of the specimens, were not provided. The only information that was provided regarding the nature of the specimens was that they were collected from non-GHB-related hanging and drowning cases, unpreserved, frozen at some time prior to analysis, and less than six months old. Were the unpreserved samples frozen immediately after collection? The nature of the specimen and its storage condition can have a significant impact on the amount of postmortem GHB production. Drowning cases often display a long postmortem interval and are frequently putrefied. It has been reported that there is an enzymatic pathway that can convert putrescine to GHB through GABA and that putrescine concentrations increase as decomposition occurs (2). Certainly postmortem production of GHB in unpreserved blood is well documented (3,4). Other studies have shown that GHB can be produced in brain specimens (5) and cerebrospinal fluid (6), especially when stored at room temperature. However, GHB production has not been observed in urine (3,4). The observations by Elliott appear to contradict these earlier findings and could have significant impact in the interpretation of postmortem GHB cases. Unfortunately, a study on the effect of putrefaction of urine in situ and possible postmortem GHB production has not been performed.

Specimen preservation also plays an important role in reducing further postmortem GHB production after specimen collection. Stephens et al. (7) found that GHB concentrations increased by 50% in the absence of sodium fluoride even when refrigerated and by 100% at room temperature. GHB concentrations reached 433 mg/L in that study. However, this was a contrived event in which the blood GHB concentration was achieved in vitro in a specimen specifically stored at 25êC for 40 days without a preservative and not a realistic situation as suggested by Elliott (1). In two sets of preserved postmortem blood specimens stored at 25êC and 4êC for 60 days, Stephens et al. (7) measured peak concentrations of postmortem GHB of 65 mg/L and 76 mg/L, respectively (n = 26). In unpreserved urine analyzed from these same cases when available (n = 17), a peak GHB concentration of 9.5 mg/L was measured. The concentrations of GHB in postmortem blood produced during the postmortem interval, though toxicologically significant, have not been reported as high as 433 mg/L. In 96 cases with a postmortem interval between autopsy and analysis of 13 to 71 days with storage at 4êC, Anderson and Kuwahara (3) found GHB concentrations ranging from 1.6 to 36 mg/mL in heart blood (preserved with NaF) and 1.7 to 48 mg/mL in femoral blood (preserved with NaF and potassium oxalate). The maximum GHB concentration in unpreserved urine was only 14 mg/mL. Fieler et al. (4) found GHB concentrations ranging from 0 to 168 mg/mL in postmortem blood, but the average concentration was only 25 mg/mL (n = 20). In eight of these cases where urine was available, no GHB was detected (limit of detection 1 mg/mL).

The author is correct that postmortem GHB concentrations should be interpreted with caution; however, the extremely high urine GHB concentrations reported by Elliott (1) are not in agreement with the results of prior studies. Elliott’s (1) findings require clarification and further evaluation including additional studies in this matrix in situ and in vitro with case history, storage conditions and analysis interval clearly defined. In conclusion, knowledge of the condition and storage history of a given specimen along with reliable case history is of utmost importance when interpretation of GHB concentration is an issue.

Laureen J. Marinetti1,2, Daniel S. Isenschmid1,2, Bradford R. Hepler1,2, and Randall L. Commissaris2
1Wayne County Medical Examiner’s Office, Detroit, Michigan
2Wayne State University, School of Pharmacy and Allied Health Professions– Department of Pharmaceutical Sciences, Detroit, Michigan

References

1. S. Elliott. The presence of gamma-hydroxybutyric acid (GHB) in postmortem biological fluids. J. Anal. Toxicol. 25: 152 (2001).
2. L. Marinetti. Gamma-hydroxybutyric acid and its analogues, gamma-butyrolactone and 1,4 butanediol. In Detection and Pharmacology of Benzodiazepines and GHB, S.J. Salamone, Ed. Humana Press, Totowa, NJ, in press.
3. D.T. Anderson and T. Kuwahara. Endogenous gamma hydroxybutyrate (GHB) concentrations in postmortem specimens. Presented at the combined meeting of CAT/NWAFS/SWAFS/SAT, Las Vegas, NV, November 7, 1997.
4. E.L. Fieler, D.E. Coleman, and R.C. Baselt. g-Hydroxybutyrate concentrations in pre- and postmortem blood and urine. Clin. Chem. 44(3): 692 (1998).
   J.D. Doherty, S.E. Hattox, O.C. Snead, and R.H. Roth. Identification of endogenous g-hydroxybutyrate in human and bovine brain and its regional distribution in human, Guinea pig and Rhesus monkey brain. J. Pharmacol. Exp. Ther. 207(1): 130–139 (1978).
5. 6O.C. Snead, III, G.B. Brown, and R.B. Morawetz. Concentration of gamma-hydroxybutyric acid in ventricular and lumbar cerebrospinal fluid. N. Engl. J. Med. 304(2): 93–95 (1981).
6. B.G. Stephens, D.E. Coleman, and R.C. Baselt. In vitro stability of endogenous gamma-hydroxybutyrate in postmortem blood. J. Forensic Sci. 44(1): 231 (1999).The author’s reply:

I welcome the response from Marinetti and co-workers to my previous preliminary investigation and their further review of the limited published literature.

As a result of this communication, I have provided further detail regarding the storage conditions and time periods pertaining to each case. In all cases, the time delay between death certification and autopsy was less than 4 days. Case 1 was analyzed 6 months following initial receipt having been stored at –20°C during this time. Cases 2, 4, 5, 6, 7, and 13 were analyzed within 52 days of receipt, and Cases 3 and 8–12 were analyzed within 14 days of receipt; all of the samples were stored at 4°C for 4 days and then at –20°C for the remaining time prior to analysis. There did not appear to be any obvious correlation between such conditions and resultant blood/urine GHB concentration.

With regard the nature of specimens, Case 5 involved extensively putrefied specimens and was the only case investigated where no GHB was detected in either blood or urine. No other cases exhibited the onset of putrefaction, and no putrefactive compounds were detected during routine toxicological analysis.

Marinetti and co-workers provide evidence from other authors regarding the detection of GHB in postmortem urine. There is insufficient information presented in the original articles to directly compare the concentrations obtained in these various studies and those obtained in my published preliminary study. Nevertheless, such data further support my findings with regard the presence of GHB in both blood and urine in postmortem biological fluid and the subsequent interpretative implications in fatalities, to which end the purpose of my published letter was to highlight this phenomenon in order to avoid any misinterpretation of the analytical data regarding GHB.

Finally, additional extensive studies involving the investigation of the presence of GHB in both postmortem and in life specimens are in progress and are expected to be published in due course.

Simon Elliott
Regional Toxicology Laboratory
City Hospital NHS Trust
Dudley Road
Birminham, U.K., B18 7QH

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