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Unexpected Changes in Urinary Catecholamines and Vanillylmandelic Acid following Rape Assault NORMAN *Department

ENDE,*

SHELDON B. GERTNER,~

AND

BARBARA

SOCHA

of Pathology, and TDepartment of Pharmacology, New Jersey School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103-2757

Medical

Although psychological changes are recognized to occur in rape assault survivors there is no information on the biochemical changes in these victims. This study compares urinary catecholamines and metabolites in 17 rape victims to two female control groups (one of which engaged in normal sexual intercourse and the other did not). We found, in the rape victims, unexpected changes in the excretion pattern of catecholamines and metabolites as compared to the various control groups. The most significant difference was the dramatic increase in urinary conjugated dopamine (P < 0.01) in the rape victims which remained elevated for over 24 hr. Urinary vanillylmandelic acid (VMA) rose significantly in rape assault victims when compared to the normal control group. The VMA levels in rape victims were significantly lower, however, than in the women who had undergone (normal) sexual intercourse (P < 0.01). Urinary free epinephrine showed a marked decline and remained depressed for over 24 hr in the rape assault victims (P < 0.01) compared to normal controls. Some possible reasons for these patterns in catecholamines and metabolite excretion are suggested. These changes may be of importance in the poststress syndrome that occurs following the rape assault. In summary, a different profile of catecholamine and metabolite excretion patterns was found in rape compared to normal sexual intercourse. The enhanced dopamine excretion is contrary to the expected change of enhanced epinephrine secretion in severe stress. o 1990 Academic PWSS, hc.

Recently while investigating cases of rape assault, we found that many of these female victims showed unusually high levels of urinary creatinine unrelated to the urine’s osmolarity (Ende, Gertner, Hwang, and Socha, 1987). In this paper we present further studies on the amounts of free and conjugated catecholamines as well as vanillylmandelic acid (VMA) present in the urine of rape assault victims. Since there are no published data on the urinary catecholamine changes present in rape assault victims, these data should prove useful. They suggest some unusual changes occurring under these conditions of severe stress.

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0 1990 by Academic Press, Inc. of reproduction in any form reserved

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METHODS Rape Assault Victims The rape assault victims consisted of 17 patients, mean age 27.1 & 8.1 years, SD, who presented at the emergency room of one of the University affiliated hospitals for treatment of rape assault. Specimens of urine were collected and acidified to pH 3.0 by adding 6 N HCl. The specimens were immediately frozen at -20°C and transferred frozen to -50°C where they were kept until studied. The victims were questioned as to the time of the rape assault and whether weapons were used and were examined for physical evidence of trauma. Controls Controls consisted of two groups. One group, “normal control,” consisted of eight normal female technicians and laboratory workers mean age 33.5 ? 6.4 years SD. Their urines were collected at the medical center during the working day and they had not received any drug or medication for 1 week prior to collection of the urine sample. The specimens after voiding were acidified with 6 N HCl to pH 3 and immediately frozen at -50°C until analysis was undertaken. For additional controls we were fortunate in obtaining urine samples from five normal female volunteers, mean age 29.8 + 2.8 years, SD, before and after sexual intercourse. These individuals were not taking any medication. The volunteers received appropriate containers and the specimens were collected in the privacy of their homes. The urines were collected and acidified to pH 3.0 by the volunteers themselves and kept at - 20°C. The samples were frozen, brought to the laboratory, and stored thereafter at -50°C. The samples marked Post are the urines obtained 1 hr postintercourse and the samples marked Pre are the urines obtained 1 hr before intercourse from the same females. Catecholamine Determination The procedure developed by Riggin and Kissinger (1977) was followed. To determine free catecholamines, 5 ml of urine and 50 ~1 of internal standard were added (dihydroxybenzylamine HBr, 15.8 mg/lOO ml) to a 25-ml beaker with 15 ml of 0.1 M (pH 7) phosphate buffer. Extraction of free catecholamines by the column occurs at pH 6.5, which is critical. Adjustment was done with 3 M NaOH. The beaker contents were poured on to a minicolumn containing a weak cation exchange resin (Biorex 70, Biorad Labs, Richmond, CA) and the column was washed with 10 ml water. The catecholamines were adsorbed and then 1.3 ml of 0.7 M H,SO, was added to the column. The catecholamines were then eluted from the column by adding 4 ml of 2 M (NH&SO, following the procedure of the authors.

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To determine conjugated catecholamines, 5.0 ml of urine was added to a 12-ml centrifuge; 200 ~1 of 12 M HCl was added and the solution was placed in a water bath (SS-9Y’C) for 30 min. After cooling the solution was transferred to a 30-ml beaker and 50 ~1 internal standard solution was added plus 20 ml of 0.1 M phosphate buffer. The pH was adjusted to 6.5 with the addition of 3 M NaOH and the procedure followed as before. The HPLC column used was a C-18 (5-pm column, 250 x 4 mm). The mobile phase was 91%, 0.1 M monochloracetate buffer (pH 3.0) containing 2 mM Na,-EDTA plus 300 mg/liter sodium octyl sulfate : 9% acetonitrile. Electrochemical detection was done with a glassy carbon electrode held at a potential +650 mV. Osmolality

The osmolality pressure method

of the urine samples was determined (Webster, 1985).

by the vapor

VMA

To measure VMA concentrations in the urine, the kit developed by Whale Scientific Co. (Commerce City, CO) was utilized (Forman, 1978). Statistical

Analysis

The analysis of variance and the Newman-Keuls test (Tallarida and Murray, 1987) were performed comparing each group to every other group. The level of P < 0.05 was considered statistically significant. Calculations

In previous publications we found that rape assault resulted in increased creatinine output in the urine which was unrelated to the osmolality (Ende et al., 1987). Due to this finding, we calculated the urinary catecholamines as rig/ml urine. The results are therefore reported as rig/ml of urine, but we also did computations on the basis of milliosmoles to convince ourselves that the changes in output were not the results of water dilutional changes. These computations are not given, but showed similar statistical results. In addition, we calculated the values, as one usually does, per milligram creatinine. The results were similar to what we report in Table 1 with similar statistical significance but are not given. RESULTS Of the 17 cases of rape assault there were 10 cases who were seen in the emergency room less than 8 hr after the assault. There were no cases that presented themselves in the emergency room between 8 and 24 hr after the assault. Four cases were seen in the emergency room at 24 hr

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66

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SOCHA

or greater (up to 72 hr) after the assaults. In the remaining three cases, time of assault was not specified. The results of urinary catecholamine determinations are given in Table 1. The most notable change was in conjugated dopamine. There was a significant increase in conjugated dopamine in the urines of the 17 rape victims as compared to the normal controls (P < 0.01). The conjugated dopamine was almost four times higher in rape victims as in normal females. The values in rape victims, however, were not significantly different from the (normal) I-hr postintercourse values. Conjugated dopamine, however, in the specimens collected from the four rape victims 24 hr after the assault were significantly elevated over the normal control group (P < 0.01) as well as elevated significantly over women who had engaged in normal sexual intercourse (P < 0.05) (Table 1). Free epinephrine dropped in all the various groups to well below the level of the control group and was significant (P < 0.01). In addition, the free dopamine level 24 hr after the rape assault was significantly lower than in the I-hr postintercourse group (P < 0.05). Conjugated epinephrine was also significantly lower in the rape victims than in the controls (P < 0.05). Of interest, was the significant increase in this value in the postintercourse group compared to the preintercourse group (P < 0.05). The VMA levels in the urine of the rape victims were very similar to those of the I-hr preintercourse controls. When compared to the VMA levels in our normal controls the levels were significantly higher P < 0.05 in the rape victims. In addition, and of greater significance, when compared to the values found in the (normal) postintercourse females, the VMA levels were lower (P < 0.01). Thus, in rape victims the amounts of urinary VMA were significantly higher compared to normal women, but were significantly lower than in women who had just engaged in (normal) sexual intercourse. This was further substantiated when we compared 11 additional pre- and postintercourse volunteers versus rape victims. These additional individuals are not included in Table 1 because they lacked the complete catecholamine data (only VMA determinations were performed on their urines). Evidence of trauma in the rape assault group was inadequate to correlate with the catecholamine changes. There was the impression that the type of weapon used by the assailant, viz., the gun, in three assaults, appeared to have the most profound effect in increasing dopamine excretion, but there were too few victims for statistical validity. DISCUSSION

Under stress or exercise, it would be expected that total urinary catecholamines would rise. Studies by other investigators have shown increases in urinary dopamine, epinephrine, and norepinephrine with ex-

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ercise or psychological stress (Bolshakova, 1976; Elmadjian, Hope, and Lamson, 1957; Fibiger and Singer, 1984; McKenzie and Fiorica, 1967; Snider, 1983; Sudoh, 1971; Wroblewski and Markiewicz, 1973). For example, there is the well-known immediate rise in catecholamine levels postexercise (Knight and Wu, 1987). There are some additional reports of a rise in serum dopamine levels and subsequent excretion following physical and mental stress (Fibiger and Singer, 1984; Snider, 1983). We were unable to find published information on urinary catecholamines in rape victims or in individuals who underwent similar severe stress. Urinary determinations have their shortcomings. It would have been extremely desirable to obtain blood samples from these victims, but this was not possible because of the victims’ disturbed psychological state and their inability to cooperate with such a study. Our most obvious finding in this study of rape victims was the rise in urinary conjugated dopamine which remained elevated for over 24 hr after the assault. This was coupled with a decrease in free epinephrine. These findings were totally unexpected and were identical regardless of the method of calculation. (i.e., per milligram or per milliosmole creatinine) . The explanation for the rise in conjugated dopamine is not clear. Animals undergoing inescapable stress have a marked depletion in catecholamines such as norepinephrine and dopamine in certain hypothalamic nuclei of the brain (DeSouza and Van Loon, 1986; Palkovits, Brownstein, Kizer, Saavedra, and Kopin, 1976). It is difficult to say whether this would be reffected in noticeably enhanced excretion of these amines, since these nuclei comprise such a small percentage of the brain matter. Thus, the possibility of these nuclei being the origin of the enhanced conjugated dopamine levels remains unlikely. On the other hand, the adrenal medulla is the largest storehouse and factory for catecholamine synthesis in the body. Therefore, a more logical explanation would be that whatever catecholamines have already been synthesized in situ by the adrenal medulla are mobilized under activation of the sympathetic nervous system under this severe stress. Dopamine, which is normally secreted in small amounts, as well as existing epinephrine (the major catecholamine) pours out. Furthermore, epinephrine synthesis under these conditions would decrease because insufficient dopamine remains in the gland to be synthesized into epinephrine. There is therefore a subsequent fall in the excretion of free epinephrine. Catecholamine synthesis increases substantially and the rape assault victims continue to pour out dopamine into the bloodstream which is quickly conjugated to the sulfate. The levels of dopamine remain at a significantly elevated level for 24 hr after the attack. These results appear to corroborate findings described in the review on dopamine by Snider and Kuchel (1983). The catecholamine changes described in this paper may

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have a relationship to the clinical changes noted in the postrape stress syndrome (Troncale, 1983; Steketee, 1987). This unusual situation of having mainly dopamine secreted by the adrenal and little, if any, epinephrine may have significant behavioral ramifications and requires additional investigations since so little is known about the behavioral effects of dopamine other than its cardiostimulatory and renal actions. The difference in epinephrine noted in the I-hr preintercourse sample from the normal controls may be related to the anticipation of sex. It is of interest that the only significant difference in osmolality was in the group sampled less than 8 hr postrape,, which was lower than controls and might indicate a salt loss during this period. We computed ng catecholamines/mosm to prove to ourselves that water dilutional or concentration changes in our samples were not responsible for altering the catecholamine levels. Thus, the amounts of urinary catecholamines were not related to the osmality of the urine. The statistical validity was similar to the data reported. From previous studies we have found that rape assault victims put out large quantities of creatinine similar to that seen in combat injuries and other types of trauma (Ende et al., 1987; Schiller, Long, and Blakemore, 1979; Frawley, Artz, and Howard, 1955). This unusual loss of creatinine in the urine reported in battle casualties has no ready explanation in the publications cited, but appears to be unrelated to the amount of muscle damage (Frawley et al., 1955). Due to our findings in rape victims, we believed it unreliable to report catecholamine data of the rape assault victims as urinary excretion per milligram of creatinine. Therefore, as previously noted, we report our data as rig/ml of urine. We were surprised to note that when the data were computed per milligram creatinine, similar statistical validity resulted. This profile of increases in conjugated dopamine, coupled with the small rise in VMA, the decrease in epinephrine, plus the increase in creatinine found previously, deserves further study to determine if some of these findings are unique to rape victims or would appear under other severe inescapable stresses. The profile is different from what occurs following normal sex. In any case, the evidence shows that the extremely severe stress of rape, which activated the sympathetic nervous system, did not cause the expected urinary elevation in epinephrine, norepinephrine, or metabolites. Unfortunately, since we had no advance knowledge of enhanced dopamine secretion, we did not study homovanillic acid levels. Considering the changes in dopamine that we found, one might expect to find increases in urinary homovanillic acid, which is the terminal metabolite for dopamine. The findings in rape victims suggests that mainly the adrenal medulla is stimulated since norepinephrine excretion does not change significantly. There is additional evidence from others that sympathetic nerve

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function also is affected in severe stress (Snider and Kuchel, 1983) which would be reflected in enhanced VMA levels. This enhanced dopamine excretion could not have come from sympathetic nerves since norepinephrine levels were unaffected. Thus, the enhanced dopamine excretion from the adrenal is contrary to the established concept of enhanced epinephrine secretion in severe stress. ACKNOWLEDGMENTS The authors thank Dr. Elizabeth Gerges for carrying out the catecholamine determinations by HPLC and Dr. S. G. Hwang for the VMA determinations. Research was supported in part by the Abraham S. Ende Research Foundation.

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Forman, D. T. (1978). Measurement of vanillylmandelic acid (VMA) by column chromatography. In F. W. Sunderman (Ed.), Seminar on Clinical Pathology of Cancer of the Endocrine GIands and Target Organs, pp. 167-177. Institute for Clinical Sciences, Inc., Philadelphia. Frawley, J. P., Artz, C. P., and Howard, J. M. (1955). Muscle metabolism and catabolism in combat casualties. Arch. Surg. 71, 612-616. Knight, J. A., and Wu, J. T. (1987). Catecholamines and their metabolites: Clinical and laboratory aspects. Lab. Med. 18, 153-158. McKenzie, J. M., and Fiorica, V. (1967). Stress responses of pilots to severe weather flying. Aerospace. Med. 38, 576-580. Palkovits, M., Brownstein, M., Kizer, J. S., Saavedra, J. M., and Kopin, 1. J. (1976). Effect of stress on serotonin and tryptophan hydroxylase activity of brain nuclei. In E. Usdin, R. Kvetnansky and I. J. Kopin (Eds.), Cutecholumines and Stress, pp. 5159. Pergamon, Elmsford, NY. Riggin, R. M., and Kissinger, P. Y. (1977). Determination of catecholamines in urine by reverse-phase liquid chromatography with electrochemical detection. Anal. Chem. 49, 2109-2111. Snider, S. R. (1983). Increased circulating dopamine levels associated with exercise, stress and hypertension. Ark. Med. 40, 333-336. Snider, S. R., and Kuchel, 0. (1983). Dopamine: An important neurohormone of the sympathoadrenal system. Endocrinol. Rev. 4, 291-309. Schiller, W. R., Long, C. L., and Blakemore, W. S. (1979). Creatinine and nitrogenous excretion in seriously ill and injured patients. Surg. Gynecol. Obstet. 149, 561-566. Steketee, G. (1987). Foa, and EB Rape victims: Post-traumatic stress responses and their treatment. J. Anx. Dis. 1, 69-86.

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Sudoh, A. (1971). Urinary excretion of catecholamines in various situations. Bull. Tokyo Med. Dent. Univ. 18, 295-310. Tallarida, R., and Murray, R. (1987). Procedure 36 Newman-Keuls Test. Manual of Pharmacologic Calculations with Computer Programs, 2nd ed. pp. 121-125. SpringerVerlag, New York. Troncale, J. A. (1983). Victims of terrorism: The physical, psychological and spiritualexistential effects. J. Med. Assoc. State Ala. 52, 49-53. Webster, H. L. (1985). Osmolality of fluids. In P. N. Cheremisinoff and R. P. Ouellette (Eds.), Biotechnology Applications and Research, pp. 607-662. Techomic, Lancaster, PA. Wroblewski, T. E., and Markiewicz, L. (1973). Excretion of catecholamines in urine under conditions of emotional stress (shocking movies). Int. Z. Angew. Physiol. Einschl. 31, 327-331.