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transplantation may decrease cardiac morbidity for these patients. Yet, at a time when medical costs are of increasing concern, there is a growing unwillingness on the part of insurers to reimburse the cost of pretransplant coronary angiography. Many diabetic transplant recipients die prematurely from undiagnosed coronary artery disease, resulting in financial loss and allograft waste. We can conclude that, when insulin-dependent diabetic patients being assessed as candidates for renal transplantation are found to have symptomless coronary artery stenoses greater than 75%, revascularisation may decrease the incidence of myocardial infarction. This conclusion should not be extended to insulin-dependent diabetic patients without advanced chronic renal disease or to non-insulin-dependent diabetic patients. The results should be interpreted with caution. Most of the study patients had juvenile-onset diabetes, and they may not be typical of diabetic patients seen at other transplant centres. Our results suggest that a larger multicentre trial is needed.

CE, Schwartz JS, Sutherland DR, et al. Predictive value of thallium stress testing for coronary and cardiovascular events in uremic diabetic patients before renal transplantation. Am J Surg 1983; 146: 331-35. 17. Subramanian UB, Bowles MJ, Davies AB, Raftery EB. Calcium channel blockade as primary therapy for stable angina pectoris: a double blind placebo-controlled comparison of verapamil and propranolol. Am J Cardiol 1981; 48: 797-803. 18. Rosa RM, Silva P, Young JB, et al. Adrenergic modulation of extrarenal potassium disposal. N Engl J Med 1980; 302: 431-34. 19. Castellino P, Bia MJ, DeFronzo RA. Adrenergic modulation of potassium metabolism in uremia. Kidney Int 1990; 37: 793-98. 20. Castellino P, Simonson DC, DeFronzo RA. Adrenergic modulation of potassium metabolism during exercise in normal and diabetic humans. Am J Physiol 1987; 252: E68-76. 21. The Working Group on Hypertension in Diabetes. Statement on hypertension in diabetes mellitus: final report. Arch Intern Med 1987; 147: 830-92. 16. Morrow

SHORT REPORT

We thank Ginny Hackenmiller for coordinating the patient evaluations, Kathy Mansir, Mary Conway, Nancy Mettille, and Lisa McConnell for collecting the data; David Fryd, David Homans, David Laxson, Gary Francis, and Tom Hostetter for valuable suggestions; and Deb Kramer for typing the paper.

Endogenous benzodiazepine receptor ligands in idiopathic recurring stupor

C. L. M. was supported by the Division of Research Resources grant MOI RR0041100 and National Institutes of Health Grant DKI3038. C. W. W. was supported by National Heart Lung and Blood Institute grant HL 39185.

REFERENCES 1. The United States Renal Data System 1990 Annual Data Report. National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland: August, 1990. 2. Weinrauch L, D’Elia JA, Healy RW, Gleason RE, Christlieb R, Leland OS. Asymptomatic coronary artery disease: angiographic assessment of diabetes evaluated for renal transplantation. Circulation 1978; 58: 1184-90. 3. Braun WE, Phillips DF, Vidt DG, et al. Coronary artery disease in 100 diabetics with end-stage renal failure. Transplant Proc 1984; 16: 603-07. 4. Lorber MI, Van Buren CT, Flechner SM, et al. Pre-transplant coronary arteriography for diabetic renal transplant recipients. Transplant Proc 1987; 19: 1539-41. 5. Weinrauch LA, D’Elia JA, Healy RW, et al. Asymptomatic coronary artery disease: angiography in diabetic patients before renal transplantation: relation of findings to postoperative survival. Ann Intern Med 1978; 88: 346-48. 6. Bennett WM, Kloster F, Rosch J, Barry J, Porter GA. Natural history of asymptomatic coronary arteriographic lesions in diabetic patients with end-stage renal disease. Am J Med 1978; 65: 779-84. 7. Braun WE, Phillips D, Vidt D, et al. The course of coronary artery disease in diabetics with and without renal allografts. Transplant Proc 1983; 15: 1114-19. 8. Philipson JD, Carpenter BJ, Itzkoff J, et al. Evaluation of cardiovascular risk for renal transplantation in diabetic patients. Am J Med 1986; 81: 630-34. 9. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Statist Assoc 1958; 88: 346-48. 10. Peto R, Peto J. Asymptotically efficient, rank invariant procedures. J R Statist Soc 1972; 135 (ser A): 135-207. 11. Machin D, Gardner MJ. Calculating confidence intervals for survival time analyses. BMJ 1988; 298: 1369-71. 12. Nesto RW, Phillips RT. Asymptomatic myocardial ischemia in diabetic patients. Am J Med 1986; 805: 40-47. 13. Boudreau RJ, Strony JT, DuCret RP, et al. Perfusion thallium imaging of type I diabetes patients with end-stage renal disease: comparison of oral and intravenous dipyridamole administration. Radiology 1990; 175: 103-05. 14. Marwick TH, Steinmuller DR, Underwood DA, et al. Ineffectiveness of dipyridamole SPECT thallium imaging as a screening technique for coronary artery disease in patients with end-stage renal failure. Transplantation 1990; 49: 100-03. 15. Holley JL, Fenton RA, Arthur RS. Thallium stress testing does not predict cardiovascular risk in diabetic patients with end-stage renal disease undergoing cadaveric renal transplantation. Am J Med 1991; 90: 563-70.

"Endozepines" are endogenous ligands for the benzodiazepine recognition sites on gammaaminobutyric acid A receptors in the nervous system. Idiopathic recurring stupor (IRS) is a syndrome of spontaneous stupor or coma that is with known metabolic, toxic, abnormalities but

can

be reversed

not or

associated structural

by flumazenil,

a

We measured

pure benzodiazepine antagonist. endozepine-2 and endozepine-4 by highand liquid performance chromatography in serum and fluid radioreceptor assay cerebrospinal from three patients with IRS. During episodes of stupor there was a large (up to 300-fold compared with control patients) increase of endozepine-4 content in cerebrospinal fluid and serum, but a return

concentrations between attacks. Endozepine-4 may contribute to, or be the cause of, IRS. The reasons for abnormal concentrations of endozepine in blood and brain are unknown. to

normal

Lancet 1992; 340:1002-04.

A group of endogenous ligands for the benzodiazepine binding site of the gamma-aminobutyric acid A receptor (GABAA) has been identified and named "endozepines".1 Their intrinsic action is, like that of diazepam, to potentiate GABAA receptor function by acting as positive allosteric modulators of this receptor, and they are present in the central nervous system in pharmacologically relevant concentrations.Endozepines purified from bovine;rat, and human brain are non-peptide, non-benzodiazepine substances that appear to have a quinoline core.’I

1003

-U. V V. IV ’———’———’———’———’———’———’———’———’———’———’———’———*—————’—————’————————————’ U -

0

10

20

30

40

60

50

HPLC Fraction Fig 1-HPLC chromatogram of CSF patient with IRS.

70

80

90 100

(min)

extract obtained

during ictal episodes

in

a

Bars under the absorbance profile represent endozepme activity in each HPLC fraction The pmol/ml endozepme-2 and 331 pmol/ml endozepine-4

extract contained 5

Experimental observations suggest that these substances play a part in normal central nervous system processes, such as memory and learning,2 and in pathological processes such as panic disorders3 or hepatic encephalopathy.4’s Idiopathic recurring stupor (IRS) was first described in a patient who had recurrent episodes of stupor and coma in the absence of known toxic, metabolic, or structural brain damage.6 IRS is reversed promptly by administration of the benzodiazepine antagonist flumazenil, suggesting that the drug may be blocking the actions of an endogenous ligand for the allosteric modulatory site of the GABAA receptor where benzodiazepines act. Flumazenil can also ameliorate hepatic encephalopathy/ which is associated with increased endozepines.4,5These observations led us to believe that IRS may be due to excess endozepines, and we tested this hypothesis by measuring endozepines in the cerebrospinal fluid (CSF) and serum of three patients with IRS. The three patients were all men aged 41-70 years. Their stuporous episodes consisted of obtundation progressing to stupor or coma from which they could be woken only by vigorous stimuli, if at all. No other neurological deficits were noted and the patients appeared to be asleep and breathing deeply. After 2-72 h they woke spontaneously and had no memory of the event. Computed tomography and magnetic resonance imaging of patients and all laboratory investigations (described previously6) done during the were for normal, stuporous episodes except electroencephalography, which showed a typical diffuse fast 13-14 Hz background activity that was unreactive to eye opening and stimulations. In all patients, flumazenil reversed stupor and normalised the electroencephalogram to a background 9 Hz activity reactive to eye opening. Serum was collected during ictal periods in three patients and during non-ictal periods in two and CSF was collected by lumbar puncture in a single patient during two different ictal periods and a non-ictal period. Control serum or CSF was obtained with informed consent from patients with normal mental status being evaluated for neurological diseases, including motoneuron disease, migraine headache, and multiple sclerosis. To extract endozepines, 2 ml of serum or CSF was added to 2-3 ml of chloroform and mixed vigorously for 1 min. The organic lower phase was collected and vacuum dried. The dried extract was then reconstituted with 1 ml

distilled, deionised

water containing 0-1% trifluoroacetic acid. Endozepines were purified from serum or CSF extracts by reverse-phase high-performance liquid chromatography (HPLC).1 HPLC fractions were collected and lyophilised, reconstituted with phosphate buffer (pH 7-4), and assayed for endozepines by radioreceptor assays.1 This protocol results in detection of two endozepines that are not halogenated benzodiazepines and do not resemble structurally any previous identified ligand for the benzodiazepine recognition site.1

HPLC analysis of CSF obtained during the ictal episode revealed a single ultraviolet-light-absorbing peak with a retention time identical to that of purified endozepine-4 and a large amount of endozepine-binding activity (fig 1). Endozepine-2 could be measured in the appropriate HPLC fraction but not in concentrations sufficient for detection by ultraviolet-light absorption (fig 1). Endozepines 2 and 4 were present in low concentrations in control serum (seven patients) and CFS (six patients) (fig 2), but were at the limit of detection ( > 1 pmol/ml) in some samples. Concentrations of endozepine-4 in serum (mean [SE] 419 [15] pmol/ml) or CSF (mean [range] 395 [335-454] pmol/ml) samples taken during ictal periods were up to 300-fold higher than concentrations in samples from control patients. During non-ictal periods, endozepine-4 concentrations in serum and CSF returned to control values in all three IRS patients. Endozepine-2 was increased in one non-ictal serum and one non-ictal CSF sample compared with the matched ictal samples (fig 2), but these changes were small compared with those observed for endozepine-4. A unique characteristic of all patients with IRS is rapid reversal of their encephalopathy by administration of the benzodiazepine antagonist flumazenil, which suggests that the syndrome could be caused by excess amounts of an endogenous ligand that binds to the benzodiazepine recognition site on the GABAA receptor and has actions like diazepam. Benzodiazepine-like material has been detected during ictal episodes in the serum and CSF of a patient with IRS,’ and we have now shown that this benzodiazepine-like material is endozepine-4. Importantly, the benzodiazepine-

1004

Endozepine-4

Control

Ictal

Non-ictal

Control

Ictal

Non-ictal

CSF

Serum

Endozepine-2 I

such as diazepam and its metabolite desmethyldiazepam, have been found in brain, albeit at physiologically insufficient concentrations.9 It is unlikely that the brain can synthesise these compounds, therefore their presence may be due to ingestion of contaminated food. We did not detect physiologically significant amounts of these benzodiazepines in the serum or CSF of IRS patients. Nevertheless, it is clear from biochemical studies10 and from studies of memory and learningz that endogenous ligands for benzodiazepine-recognition sites on the GABAA receptor exist, and endozepine-4 may be one of these ligands. The cause of raised concentrations of endozepine-4 is not known. It could be the result of abnormal enzymatic degradation of the substance or of a large increase in its precursor supply. Since endozepine-4 concentrations were increased in both serum and CSF, the defect is clearly not limited to the central nervous system. There was no obvious liver dysfunction in IRS patients or unusual dietary habits. Thus, IRS appears to be a disorder of excess endozepines, but the exact nature of the defect is unknown and its elucidation awaits further knowledge of endozepine metabolism. We thank Dr Rosella Avallonne for radioreceptor assays. This work was supported by a National Institute of Neurological Disease and Stroke clinical investigator award (NS01355) to J. D. R.

REFERENCES

0’

ae Control ictal

,

V

Non-ictal

Serum

v’ Control tctaf

’

Non-ictal

CSF

Fig 2-Concentrations of endozepine-2 and endozepine-4 in serum and CSF from patients with IRS and from control patients.

like actions of endozepine-4--eg, potentiation of GABAevoked chloride currents-are specifically reversed by flumazenil.1 Excess amounts of endozepine-4 were present in our patients during ictal episodes and are probably the cause of the encephalopathy. The concentration of endozepine-4 during stuporous episodes ranged from 100-650 nmol/1 and similar concentrations of benzodiazepines, such as diazepam, produce stupor.8 Since endozepine-4 is an agonist like diazepam and has the same affinity for the benzodiazepine-binding site, it is reasonable to assume that the concentrations of endozepine-4 we detected are responsible for the stupor in IRS. Endozepine-2 has properties similar to endozepine-4 but appears to be less potent than endozepine-4 (unpublished observations). Concentrations of endozepine-2 in the serum and CSF of IRS patients were not greatly altered during stupor and coma. The chemical relation between endozepines 2 and 4 is not yet known. Because IRS can be reversed by flumazenil, it is possible that compounds related structurally to diazepam contribute to the syndrome. Indeed, halogenated benzodiazepines,

1. Rothstein JD, Garland W, Puia G, Guidotti A, Weber R, Costa E. Purification and characterization of naturally occurring benzodiazepine receptor ligands. J Neurochem 1992; 58: 2102-15. 2. Izquierdo I, Medina J GABAA receptor modulation of memory: the role of endogenous benzodiazepines. Trends Neurosci 1991; 12: 260-65. 3. Nutt DJ, Glue P, Lawson C, Wilson S. Plumazenil provocation of panic attacks. Arch Gen Psychiatry 1990; 47: 917-25. 4. Olasmaa M, Rothstein JD, Guidotti A, et al. Endogenous benzodiazepine receptor ligands in human and animal hepatic encephalopathy.

J Neurochem 1990; 55:

2015-23.

KD, Szauter KM, Kaminsky-Russ K. "Endogenous" benzodiazepine activity in physiological fluids of patients with hepatic encephalopathy. Lancet 1990; 550: 81-83. 6. Tinuper P, Montagna P, Cortelli P, et al. Idiopathic recurring stupor. A case with possible involvement of the GABAergic system. Ann Neurol 1992; 31: 503-06. 7. Grimm G, Ferenci P, Katzenschlager R, et al. Improvement of hepatic encephalopathy treated with flumazenil. Lancet 1988; i: 1292-94. 8. Mennini T, Garattini S. Benzodiazepines receptor binding in vivo: pharmacokinetic and pharmacological significance. Adv Biochem Psychopharmacol 1983; 38: 189-99. 9. Unseld E, Krishna DR, Fischer C, Klotz U. Detection of desmethyldiazepam and diazepam in brain of different species and plants. Biochem Pharmacol 1989 38 2473-78. 10. Costa E, Guidotti A. Neuropeptides as cotransmitters: modulatory effects at GABAergic synapses. In: Meltzer HY, ed. Psychopharmacology: the third generation of progress. New York: Raven Press, 1987:425-35. 5. Mullen

ADDRESSES.

Department of Neurology, Johns Hopkins University, Meyer 5-119, 600 North Wolfe Street, Baltimore, Maryland 21205, USA (J. D. Rothstein, MD); Fidia-Georgetown Institute for Neuroscience, Georgetown University, Washington, DC (A. Guidotti, MD); Institute of Neurology, University of Bologna, Bologna, Italy (P. Tinuper, MD, P. Cortelli, MD, P Avoni, MD, G. Plazzi, MD, E. Lugaresi, MD, P. Montagna, MD); Preclinical Research, Pharma Division, Hoffman-La Roche, Basel, Switzerland (P. Schoch, PhD). Correspondence to Dr

and

Jeffrey

D Rothstein.