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Lithium incorporation in the fibroblasts of manic-depressives
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Lithium Incorporation in the Fibroblasts of Manic-Depressives Richard E. Breslow, George W. DeMuth, and Cipora Weiss
Lithium incorporation in cultured human skin fibroblasts was measured in a group of 10 manic-depressives and 10 controls. This system was believed to eliminate many of the sources of variability to which measurement of the lithium ratio in erythrocytes (RBCs) is subject. A fibroblast lithium ratio calculated from 1-hr lithium incorporation studies correlated highly with an in vitro RBC lithium ratio in medication-free controls. Manicdepressives and controls did not differ in lithium incorporation or fibroblast lithium ratio, leading to the conclusion that the lithium ratio is probably not a good measure for differentiating the two populations. Lithium has proved an effective agent for the treatment (Schou et al. 1954) and prevention (Davis 1976) of manic-depressive illness. It has been suggested (Mendels and Frazer 1973) that a central nervous system (CNS) membrane defect of lithium transport might relate to either the pathogenesis or the treatment responsiveness of the disorder. Although certain aspects of the cellular transport of lithium have been described at the molecular level (Ehrlich and Diamond 1979; Pandey et al. 1979a), the mode of therapeutic action of lithium as well as the underlying biological defect in manic-depressive illness remain unknown. In an effort to define a cell system that is both readily accessible in human subjects and that reflects the transport of lithium by CNS cells, we have studied the lithium distribution ratio in the cultured skin fibroblast. Fibroblasts can be readily collected from patients. They show genetic variability and have been used extensively throughout general medicine in the study of genetic disease. Because fibroblasts derive from the neural crest, they are closely related to CNS cells. This raises the possibility that membrane transport parameters in cultured fibroblasts closely parallel those of CNS cells, thus providing a valid model for the study of neuronal transport. In prior work, the red blood cell (RBC) has been considered a model for CNS membrane function in studies of lithium transport. RBC lithium concentration and plasma lithium can readily be measured at equilibrium, dividing the former by the latter to give the socalled lithium ratio. Early studies indicated that the RBC lithium ratio is increased in manic-depressives when bipolars are compared with unipolars (Frazer et al. 1978; Ramsey et al. 1979; Szentistvanyi and Janka 1979) or with healthy controls (Pandey et al. 1979a; From the Department of Psychiatry, Downstate Medical Center, Brooklyn, New York 11203. Supported in part by grant MH-36297 from the National Institute of Mental Health. Address reprint requests to: Dr. Richard E. Breslow, Department of Psychiatry, Box 1203, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203. Received April 19, 1984; revised July 6, 1984.
© 1985 Society of Biological Psychiatry
0006-3223/85/503.30
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59
Lyttkens et al. 1973) and when lithium responders are compared with nonresponders (Sacchetti et al. 1977; Flemenbaum et al. 1978). A number of studies have found a high correlation between the lithium ratio and such measures of lithium transport as the in vitro lithium ratio and Na-Li countertransport (Szentistvanyi and Janka 1979; Rybakowski et al. 1977; Pandey et al. 1978). Ostrow et al. (1978) found that the in vitro measures were significantly different in a population of manic-depressives as compared with those of healthy controls. However, currently there are an equal or greater number of reports of failure to replicate these findings for the lithium ratio, including those by Frazer et al. (1978), for bipolars versus controls, by Albrecht et al. (1976) and Demisch and Bochnik (1976), for bipolars versus unipolars, and by Rybakowski and Stryzyewski (1976) and Mendelewicz and Verbanck, (1976), for responders versus nonresponders. Even if a study shows a significant difference in mean lithium ratios between manic-depressives and normals, there is always considerable overlap between individual patients in the two populations. A number of methodological difficulties complicate these studies (Frazer et al. 1978). The RBC lithium ratio is known to be affected by prior exposure to lithium (Dunner et al. 1978; Meltzer et al. 1977; Rybakowski et al. 1978; Ehrlich and Diamond 1979). Thus, chronic lithium administration may obscure disease related abnormalities of the lithium ratio. In addition, data from patients concurrently taking other medications that affect the lithium ratio (Pandy et al. 1979b; Ostrow et al. 1980) may further confuse illness related abnormalities. Also, it is difficult to collect control data on lithium ratios because such a study involves placing nonpatients on lithium for prolonged periods of time. This difficulty has led to the development of an in vitro technique for studying lithium ratios in RBCs, but this technique still leaves the problem that lithium-treated patients have long-term changes in their RBC membranes induced by lithium that would obscure illnessrelated abnormalities. Finally, the RBC is a nongrowing, nonnucleated cell that is not closely related to the CNS cells embryologically. The purpose of the present study was to determine whether the cultured human fibroblast is a useful alternative model for studying lithium transport. We attempted to do this by comparing an in vitro fibroblast lithium ratio calculated from a lithium uptake study with RBC lithium ratios measures in vitro. Next, we used this fibroblast system to provide data relevant to the hypothesis that the lithium ratio is higher in manic-depressives than in controls. Methods The manic-depressive group consisted of 10 outpatients from the Affective Disorders Clinic of Downstate Medical Center, New York, ranging in age from 26 to 68 years (mean _+ SD 45.7 ___ 11.2). There were six women and four men in the manic-depressive sample. All subjects were white and all were normotensive. The diagnosis of manicdepressive illness was established according to the Research Diagnostic Criteria of Spitzer et al. (1981) by the treating psychiatrist; in each case it was independently corroborated by a second psychiatrist on the basis of a semistructured clinical interview. The subjects comprised the entire population of the clinic upon which both psychiatrists could agree on a bipolar I diagnosis. Patients with schizophrenic symptomatology, significant medical illness, and prior history of alcohol or drug abuse were also excluded. All subjects were in remission between episodes of illness. All were on maintenance lithium therapy with plasma lithium levels of 0.2-1.0 mEq/liter at the time of testing. Subjects were ill a
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substantial length of time at testing ranging from 7 years to 31 years with a mean length of illness of 19.3 years _+ 8.3 years. All were lithium responders during the course of their acute episodes. Control subjects were age-matched members of the university community, ranging in age from 26 to 66 years (41.8 _+ 12.2, t = 0.746, df = 18, NS). There were five women and five men in the control sample. All controls were white and all were normotensive. Controls were carefully screened to exclude present or past psychiatric illness, major medical or surgical illness, alcohol or drug abuse, and family history compatible with either schizophrenic or primary affective illness. All control subjects and patients gave written informed consent to the procedure used in accordance with established standards of the Downstate Institutional Review Board. Punch biopsies of skin (volar surface, forearm) were obtained under local anesthesia from each subject. Tissue was washed in culture medium [Eagle's Minimum Essential Medium (MEM), Flow Laboratories, Inc.] and cut into small pieces under sterile conditions for culturing by standard cell-culture techniques. Outgrowth of fibroblasts was noted after 2 wk and a monolayer formed sufficient in size for transfer to flasks with trypsin in approximately l month. After 2 weeks of additional growth, cells were seeded onto Falcon P60 dishes (approximately 5 x l05 cells per dish) and grown to confluence for lithium-incorporation experiments. Experiments were done with confluent cells during the stationary phase of cell growth in order to avoid effects of cell growth on lithium incorporation values. Preliminary studies indicated that lithium incorporation rises rapidly in medium containing 20 mM lithium chloride--much more rapidly than in the RBC system. Between 1/2 and 1 hr lithium incorporation reaches a maximum level, which persists until a point between 2 and 4 hr. Beyond that point, incorporation decreases, probably because of the effect of such high concentrations of lithium on cell viability. A 1-hr period was selected for further studies as representing a steady-state lithium level. P60 dishes were washed in saline to remove all residual MEM and fetal calf serum. They were then incubated at 37 ° C in a specially prepared incorporation medium consisting of LiCl 20 mM, NaCI 130 mM, KCI 5 raM, MgC12 l mM, CaC12 0.25 mM, Na2HPO4 5 mM, NaHEPO4 l mM, and glucose I 1. l mM. (For control samples, the 20 mM LiCl was replaced by NaCl.) Incorporation was terminated after 1 hr by removal of the LiCl incubation medium and five rapid washes with ice-cold sucrose solution (320 mM). Preliminary studies indicated that this removes essentially all of the extracellular lithium, while leaving membrane-bound and intracellular lithium intact. After drying, 2 ml of distilled water was added to each monolayer to lyse the cells. The cell layer was disrupted and detached by thorough scraping with a rubber policeman, and the fluid was drawn up for determination of cellular lithium and total protein. Lithium in the lysate was measured by atomic absorption spectrophotometry using a Perkin Elmer Model 2280 with a graphite furnace attachment. Standards were mixed using approximately the intracellular ionic content of the cell. Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Lithium uptake results were expressed as nanomoles of Li per milligram cell protein. In preliminary studies, lithium incorporation was measured at 12, 16, and 20 generations in culture with no differences found. All studies were done in cells in these early generations in culture. We believed that it would be helpful, in comparing the lithium incorporation data with data from other cell types, particularly the RBC, to calculate a lithium distribution ratio for the fibroblast. We showed that fibroblasts tend to be uniform in size with a volume
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of approximately 900 ixm3 (9.0 x 10-712,1)per cell by the use of the cell-sizing attachment of a Coulter/Counter; this value was independently confirmed by spinning down a large number of cells in medium with Co ~° EDTA added to serve as an extraeellular marker (Brading and Jones 1969). Using the l-hr lithium uptake for these cells (in nanomoles per milligram cell protein), the cell volume determined above (9 x 10-7p, l), an average determination for the protein content of a cell (2.5 x 10 4 p,g/cell), and an average concentration of Li in the supernatant (16 mM), we calculated the l-hr lithium distribution ratio or fibroblast "lithium ratio," as reported in Table 1. Comparison in vitro RBC lithium ratios were obtained for most subjects using the technique of Pandey and Davis (1977). Incorporation experiments, lithium ratio determinations, and subsequent calculation of results were performed with the investigator blind as to the identity of the subjects. All determinations reported were the results of averaging at least triplicate values for each sample. If experiments on a given subject were performed two or more times, results of each experiment were averaged to give the reported value. Statistical comparisons were made using Student's t-test and the Pearson-product moment correlation.
Table 1. In Vitro Lithium Ratios and Fibroblast Lithium Incorporation
Subject
Li level
Fibroblast l-hr Li incorp.
RBC in vitro Li ratio
(nmol/Li/p,g
Fibroblast I-hr Li
protein)
dist. ratio
code
Sex
Age
MST TSM EPH LPA SPA DIN HRO GDF SOK FFR
F M F F M F M F M F
49 40 39 55 40 27 51 48 40 68
0.7 0.2 1.0 0.8 0.9 1.0 0.8 0.8 0.2 0.2
0.41 0.36 0.48 O. 22 0.49 0.50 -0.34 -0.37
145.2 92.9 101.3 198.4 128.0 149.8 75.9 76.3 51.7 ! 19.3
2.53 1.62 1.76 3.45 2.23 2.61 1.32 1.33 0.90 2.08
45.7 11.2
0.7 0.3
0.40 0.09
113.9 43.4
1.98 0.76
66 43 26 35 28 32 46 41 51 50
----------
--0.46 0.35 0.39 0.33 0.32 0.41 0.28 0.58
97.3 115.8 69.0 72.4 111.4 75.7 70.2 89.8 90.0 157.7
1.69 2.01 1.20 1.26 1.94 1.32 1.22 1.56 1.57 2.74
41.8 12.2
---
0.39 O. 10
94.9 27.6
1.65 0.48
(mEq/liter)
Manic-depressives
M e a n SD
Controls CRF CRE BDS LIS
BDT SUL MKE HRD WSS BGR
Mean SD
M M F M F F M M F F
--
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BIOL PSYCHIATRY 1985 ;20:58-65
R . E . B r e s l o w et
al.
Results Table 1 presents the data obtained from the 10 manic-depressive and 10 control subjects. These includes the sex, age, plasma lithium level, RBC in vitro lithium ratio, fibroblast l-hr lithium incorporation, and fibroblast 1-hr lithium distribution, or "fibroblast lithium ratio." Note in particular that all manic-depressives received lithium, while no control subjects received lithium. RBC data could not be obtained for two manic-depressives and two controls, so that RBC lithium ratio values are from the eight remaining subjects in each category. The lithium ratios derived from both cell systems were compared. For the control group, there was a high degree of correspondence in lithium distribution between the two cell types (r = 0.71, df = 6, p < 0.05, two-tailed test). This comparison was nonsignificant for the manic-depressive group (r = 0.30, df = 6, NS) and for both groups combined (r = 0.14, df = 14, NS). The mean RBC lithium ratio for eight manic-depressives was 0.40 _ 0.09, whereas eight controls had a mean ratio of 0.39 +_ 0.10. There were no significant differences between the two groups (t = 1.01, df = 14). In the fibroblasts of the control population, we found a mean of 94.9 _ 27.6 nmol lithium was incorporated per milligram of cell protein. This yielded a "lithium ratio" of 1.65 +_ 0.40 for the fibroblast, approximately four times higher than for RBCs. The fibroblasts of the manic-depressive population showed a mean lithium incorporation of 113.9 --+ 43.4 nmol of lithium per milligram cell protein, equivalent to a fibroblast lithium ratio of 1.98 _ 0.76. No significant difference was found between the two groups (t = 1.16, df = 18, NS). As can be seen from Figure 1, in both the RBC and the fibroblast systems, there is considerable overlap in the data. Many fibroblast metabolic and transport properties seem to vary with age. However, this sample showed no evidence of an age effect on fibroblast lithium incorporation (r = 0.13, NS) for the pooled sample of manic-depressives and controls. The values for each group considered separately were also not significant. The RBC lithium ratio also showed no relationship with age in the pooled group (r = - 0 . 3 2 , df = 14, NS) or when manic-depressives and controls were considered separately. In the manic-depressive population, both the fibroblast and RBC lithium ratios showed little correlation with plasma lithium level (r = 0.38 in both cases, NS), age of illness onset (r = 0.45 and - 0 . 6 2 , both NS), or duration of illness (r = - 0 . 4 9 and 0.08, respectively, both NS). - 200
.6-150
o
.5-
0•0
.4
._l--
°n o
_.~ - I00
mno I• I
.2=
-50
.I c
normol controls
monic depressives
~
25
.3
monic depressives
.c o o
normol controls
E e
Figure 1. Lithium ratios and lithium incorporation in erythrocytes and fibroblasts of manic-depressives and controls. (A) RBC in vitro Li ratio. (B) Cultured fibroblast in vitro Li ratio and l-hr Li incorporation. Li incorporation is shown in right-hand scale. (ll) male; (0) female.
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Discussion Our data support the possibility that the fibroblast is a valid model for the study of lithium transport mechanisms. The cultured fibroblasts have the same genetic material as the other somatic cells of the manic-depressive subjects from which they derive but have never been exposed to lithium. Therefore, they may provide a better source of comparison with the control populations, which also had ho lithium exposure. The data show a high correlation between the fibroblast lithium ratio and the in vitro RBC ratio in lithium-free control subjects. This finding further reinforces the possibility that the lithium extrusion mechanisms function in a similar fashion in both cell types within subjects. There is a higher equilibrium ratio in the fibroblast because of the lower electrical potential inside the cell. However, it still must be explained why there is such poor correlation between the fibroblast "lithium ratio" and the RBC lithium ratio in the manic-depressive group. This may be accounted for by the fact that chronic lithium treatment gradually changes the RBC lithium ratio by a specific inhibiting effect on the Na-Li countertransport system (Meltzer et al. 1977). No such effect would occur on the fibroblasts taken from manic-depressives, since the skin fibroblasts get little exposure to lithium and are grown in vitro for a number of generations without exposure to lithium. Because there is a factor altering the lithium ratio in one cell type (RBCs) but not the other (fibroblasts), the correlation between the two measures for this population is very low (r = 0.30). The fibroblast lithium ratio in the manic-depressive population is expected to be higher than in controls because of the hypothesized genetic defect in countertransport, as Ostrow et al. (1978) demonstrated in the RBC. With less efflux of lithium from the cell, because of the higher electrochemical gradient in the fibroblast, one would expect markedly accentuated differences in lithium distribution ratios between manic-depressives and controls in this system. The data do not support this. The mean fibroblast lithium ratio in the manic-depressive population was higher than in controls, but only by 20% (1.98 vs. 1.65), and the range was so great (0.90-3.45) that the overlap is considerable (see Fig. 1), yielding a nonsignificant difference. This finding could indicate either that there is a small difference in fibroblast lithium distribution ratios between the two populations but an insufficient number of subjects have been tested for it to show up or, what appears to be more likely, that there is no difference between the two populations on this measure. In either case, the current data do not support the hypothesis that an inherited membrane transport defect in countertransport exists generally in the somatic cells of manic-depressives. Previous studies in RBCs that demonstrated differences in lithium distribution between manic-depressives and controls were done using patients taking lithium, suggesting that the differences could be an artifact of the effect of chronic lithium treatment on the countertransport system. The present study establishes the technique of fibroblast cell culture as a new tool for the investigation of lithium transport differences in psychiatric populations. This method was employed in a comparison between a manic-depressive and a normal population, making it possible to circumvent the problem that manic-depressives are usually on chronic lithium treatment, which would distort the lithium transport parameters of their RBCs. Lithium responders were not compared with nonresponders, since all our patients were lithium responders, nor were unipolars and bipolars compared, since our population consisted entirely of bipolars and of a matched group of normals. It would be reasonable to assume, however, that if any differences were obtained they would be even greater
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between manic-depressives and normals than between responders and nonresponders or unipolars and bipolars. Therefore, it was particularly striking that the overlap was so great and no significant differences were found, leading to the conclusion that the lithium ratio is probably not a good measure for differentiating the two populations. This is not an unexpected result, as the current measure is a gross general measure of the overall equilibrium distribution inside and outside the cell. Further work is certainly indicated to look for more subtle differences in underlying transport mechanisms between the two populations. The use of specific inhibitors of transport pathways and special conditions of studying transport should make possible a much better delineation of the different pathways involved in establishing the lithium distribution ratio in the fibroblast. Perhaps the measurement of one of these pathways might reveal much greater differences in the two populations. This would enable us to study the etiology or pathogenesis of manic-depressive illness in terms of the specific membrane transport defect involved.
References Albrecht V, Muller-Oerlinghausen B (1976): Zur Klinischen bedeutung der intraerythrozytaren lithium konzentration: Ergebnisse einer katanmestischen Studie. Arzneim.-Forsch. 26:1145-1147. Brading A, Jones A (1969): Distribution and kinetics of Co-EDTA in smooth muscle, and its use as an extracellular marker. J Physiol (Lond) 200:387-392. Davis, JM (1976): Maintenance therapy in psychiatry: II, the affective disorders. Am J Psychiatry 133:1-13. Demisch V, Bochnik HJ (1976): Zur verbesserung der lithium prophylaxe endogen phasicher psychosen: Aspekte der parallelen lithium bestimmung im serum und in erythrozyten. Arzneim.Forsch. 26:1149-1 ! 51. Dunner DL, Meltzer HL, Fieve RR ( 1978): Clinical correlates of the lithium pump. Am J Psychiatry 135:1062-1064. Ehrlich BE, Diamond JM (1979): Lithium fluxes in human erythrocytes. Am J Psychiatry 237:C 102--C110. Flemenbaum A, Weddige R, Miller J (1978): Lithium erythrocyte/plasma ratio as a predictor of response. Am J Psychiatry 135:336-338. Frazer A, Mendels J, Brunswick D, et al (1978): Erythrocyte concentration of the lithium ion: Clinical correlates and mechanisms of action. Am J Psychiatry 135:1065-1069. Lowry OH, Rosenbrough NJ, Farr AL, et al (1951): Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275. Lyttkens L, Soderberg U, Wetterberg L (1973): Increased lithium erythrocyte/plasma ratio in manic depressive psychosis. Lancet 1:40. Meltzer HL, Kassir S, Dunner DL (1977): Repression of a lithium pump as a consequence of lithium ingestion by manic depressive subjects. Psychopharmacology 113-118. Mendels J, Frazer A (1973): lntracellular lithium concentration and clinical response: Toward a membrane theory of depression. J Psychiat Res 10:9-18. Mendelewicz J, Verbanck P (1977): Lithium ratio and clinical response in manic depressive illness. Lancet 1:41. Ostrow DG, Southam AS, Davis JM (1980): Lithium--drug interactions altering the intracellular lithium level: An in vitro study. Biol Psychiatry 15:723-739. Ostrow DG, Pandey GN, Davis JM, et al (1978): A heritable disorder of lithium transport in erythrocytes of a subpopulation of manic depressive patients. Am J Psychiatry 135:1070--1078. Pandey GN, Baker J, Chang S, et al (1978): Prediction of in vivo red cell/plasma Li ratios by in vitro methods. Clin Pharmacol Ther 24:343-349.
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Pandey GN, Davis JM (1979): A procedure for the determination of Li ratio in vitro of human erythrocytes. In Cooper TB, Gershon S (eds), Lithium: Controversies and Unresolved Issues, Amsterdam: Excerpta Medica, pp 969-975. Pandey GN, Dorus E, Davis JM, et al (1979a): Lithium transport in human red blood cells. Arch Gen Psychiatry 36:902-908. Pandey GN, Goel I, Davis, JM (1979b): Effect of neuroleptic drugs on lithium uptake by the human erythrocyte. Clin Pharmacol Ther 26:96-102. Ramsey TA, Frazer A, Mendels J, et al (1979): The erythrocyte lithium-plasma lithium ratio in patients with primary affective disorder. Arch Gen Psychiatry 36:457-461. Rybakowski J, Frazer A, Mendels J, et al (1978): Erythrocyte accumulation of the lithium ion in control subjects and patients with primary affective disorder. Commun Psychopharmacol 2:99-104. Rybakowski J, Frazer A, Mendels J, et al (1977): Prediction of the lithium ratio in man by means of an in vitro test. Clin Pharmacol Ther 22:465-469. Rybakowski J, Strzyzewski W (1976): Red blood cell lithium index and long term maintenance treatment. Lancet 1:1408-1409. Sacchetti E, Bottinelli S, Bellodi L, et ai (1977): Erythrocyte/plasma lithium ratios. Lancet 1:908. Schou M, Juel-Nielsen N, Stromgren E, et al (1954): The treatment of manic psychoses by the administration of lithium salts. J Neurol Neurosurg Psychiatry 17:250-260. Spitzer RL, Endicott J, Robins E (1981): Research Diagnostic Criteria for a Selected Group of Functional Disorders, 2rid ed. New York: New York Biometrics Research Division, New York State Psychiatric Institute. Szentistvanyi I, Janka Z (1979): Correlation between the lithium ratio and Na-dependent Li transport of red blood cells during lithium prophylaxis. Biol Psychiatry 14:973-977.
BIOL PSYCHIATRY 1985;20:58--65
Lithium Incorporation in the Fibroblasts of Manic-Depressives Richard E. Breslow, George W. DeMuth, and Cipora Weiss
Lithium incorporation in cultured human skin fibroblasts was measured in a group of 10 manic-depressives and 10 controls. This system was believed to eliminate many of the sources of variability to which measurement of the lithium ratio in erythrocytes (RBCs) is subject. A fibroblast lithium ratio calculated from 1-hr lithium incorporation studies correlated highly with an in vitro RBC lithium ratio in medication-free controls. Manicdepressives and controls did not differ in lithium incorporation or fibroblast lithium ratio, leading to the conclusion that the lithium ratio is probably not a good measure for differentiating the two populations. Lithium has proved an effective agent for the treatment (Schou et al. 1954) and prevention (Davis 1976) of manic-depressive illness. It has been suggested (Mendels and Frazer 1973) that a central nervous system (CNS) membrane defect of lithium transport might relate to either the pathogenesis or the treatment responsiveness of the disorder. Although certain aspects of the cellular transport of lithium have been described at the molecular level (Ehrlich and Diamond 1979; Pandey et al. 1979a), the mode of therapeutic action of lithium as well as the underlying biological defect in manic-depressive illness remain unknown. In an effort to define a cell system that is both readily accessible in human subjects and that reflects the transport of lithium by CNS cells, we have studied the lithium distribution ratio in the cultured skin fibroblast. Fibroblasts can be readily collected from patients. They show genetic variability and have been used extensively throughout general medicine in the study of genetic disease. Because fibroblasts derive from the neural crest, they are closely related to CNS cells. This raises the possibility that membrane transport parameters in cultured fibroblasts closely parallel those of CNS cells, thus providing a valid model for the study of neuronal transport. In prior work, the red blood cell (RBC) has been considered a model for CNS membrane function in studies of lithium transport. RBC lithium concentration and plasma lithium can readily be measured at equilibrium, dividing the former by the latter to give the socalled lithium ratio. Early studies indicated that the RBC lithium ratio is increased in manic-depressives when bipolars are compared with unipolars (Frazer et al. 1978; Ramsey et al. 1979; Szentistvanyi and Janka 1979) or with healthy controls (Pandey et al. 1979a; From the Department of Psychiatry, Downstate Medical Center, Brooklyn, New York 11203. Supported in part by grant MH-36297 from the National Institute of Mental Health. Address reprint requests to: Dr. Richard E. Breslow, Department of Psychiatry, Box 1203, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203. Received April 19, 1984; revised July 6, 1984.
© 1985 Society of Biological Psychiatry
0006-3223/85/503.30
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59
Lyttkens et al. 1973) and when lithium responders are compared with nonresponders (Sacchetti et al. 1977; Flemenbaum et al. 1978). A number of studies have found a high correlation between the lithium ratio and such measures of lithium transport as the in vitro lithium ratio and Na-Li countertransport (Szentistvanyi and Janka 1979; Rybakowski et al. 1977; Pandey et al. 1978). Ostrow et al. (1978) found that the in vitro measures were significantly different in a population of manic-depressives as compared with those of healthy controls. However, currently there are an equal or greater number of reports of failure to replicate these findings for the lithium ratio, including those by Frazer et al. (1978), for bipolars versus controls, by Albrecht et al. (1976) and Demisch and Bochnik (1976), for bipolars versus unipolars, and by Rybakowski and Stryzyewski (1976) and Mendelewicz and Verbanck, (1976), for responders versus nonresponders. Even if a study shows a significant difference in mean lithium ratios between manic-depressives and normals, there is always considerable overlap between individual patients in the two populations. A number of methodological difficulties complicate these studies (Frazer et al. 1978). The RBC lithium ratio is known to be affected by prior exposure to lithium (Dunner et al. 1978; Meltzer et al. 1977; Rybakowski et al. 1978; Ehrlich and Diamond 1979). Thus, chronic lithium administration may obscure disease related abnormalities of the lithium ratio. In addition, data from patients concurrently taking other medications that affect the lithium ratio (Pandy et al. 1979b; Ostrow et al. 1980) may further confuse illness related abnormalities. Also, it is difficult to collect control data on lithium ratios because such a study involves placing nonpatients on lithium for prolonged periods of time. This difficulty has led to the development of an in vitro technique for studying lithium ratios in RBCs, but this technique still leaves the problem that lithium-treated patients have long-term changes in their RBC membranes induced by lithium that would obscure illnessrelated abnormalities. Finally, the RBC is a nongrowing, nonnucleated cell that is not closely related to the CNS cells embryologically. The purpose of the present study was to determine whether the cultured human fibroblast is a useful alternative model for studying lithium transport. We attempted to do this by comparing an in vitro fibroblast lithium ratio calculated from a lithium uptake study with RBC lithium ratios measures in vitro. Next, we used this fibroblast system to provide data relevant to the hypothesis that the lithium ratio is higher in manic-depressives than in controls. Methods The manic-depressive group consisted of 10 outpatients from the Affective Disorders Clinic of Downstate Medical Center, New York, ranging in age from 26 to 68 years (mean _+ SD 45.7 ___ 11.2). There were six women and four men in the manic-depressive sample. All subjects were white and all were normotensive. The diagnosis of manicdepressive illness was established according to the Research Diagnostic Criteria of Spitzer et al. (1981) by the treating psychiatrist; in each case it was independently corroborated by a second psychiatrist on the basis of a semistructured clinical interview. The subjects comprised the entire population of the clinic upon which both psychiatrists could agree on a bipolar I diagnosis. Patients with schizophrenic symptomatology, significant medical illness, and prior history of alcohol or drug abuse were also excluded. All subjects were in remission between episodes of illness. All were on maintenance lithium therapy with plasma lithium levels of 0.2-1.0 mEq/liter at the time of testing. Subjects were ill a
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substantial length of time at testing ranging from 7 years to 31 years with a mean length of illness of 19.3 years _+ 8.3 years. All were lithium responders during the course of their acute episodes. Control subjects were age-matched members of the university community, ranging in age from 26 to 66 years (41.8 _+ 12.2, t = 0.746, df = 18, NS). There were five women and five men in the control sample. All controls were white and all were normotensive. Controls were carefully screened to exclude present or past psychiatric illness, major medical or surgical illness, alcohol or drug abuse, and family history compatible with either schizophrenic or primary affective illness. All control subjects and patients gave written informed consent to the procedure used in accordance with established standards of the Downstate Institutional Review Board. Punch biopsies of skin (volar surface, forearm) were obtained under local anesthesia from each subject. Tissue was washed in culture medium [Eagle's Minimum Essential Medium (MEM), Flow Laboratories, Inc.] and cut into small pieces under sterile conditions for culturing by standard cell-culture techniques. Outgrowth of fibroblasts was noted after 2 wk and a monolayer formed sufficient in size for transfer to flasks with trypsin in approximately l month. After 2 weeks of additional growth, cells were seeded onto Falcon P60 dishes (approximately 5 x l05 cells per dish) and grown to confluence for lithium-incorporation experiments. Experiments were done with confluent cells during the stationary phase of cell growth in order to avoid effects of cell growth on lithium incorporation values. Preliminary studies indicated that lithium incorporation rises rapidly in medium containing 20 mM lithium chloride--much more rapidly than in the RBC system. Between 1/2 and 1 hr lithium incorporation reaches a maximum level, which persists until a point between 2 and 4 hr. Beyond that point, incorporation decreases, probably because of the effect of such high concentrations of lithium on cell viability. A 1-hr period was selected for further studies as representing a steady-state lithium level. P60 dishes were washed in saline to remove all residual MEM and fetal calf serum. They were then incubated at 37 ° C in a specially prepared incorporation medium consisting of LiCl 20 mM, NaCI 130 mM, KCI 5 raM, MgC12 l mM, CaC12 0.25 mM, Na2HPO4 5 mM, NaHEPO4 l mM, and glucose I 1. l mM. (For control samples, the 20 mM LiCl was replaced by NaCl.) Incorporation was terminated after 1 hr by removal of the LiCl incubation medium and five rapid washes with ice-cold sucrose solution (320 mM). Preliminary studies indicated that this removes essentially all of the extracellular lithium, while leaving membrane-bound and intracellular lithium intact. After drying, 2 ml of distilled water was added to each monolayer to lyse the cells. The cell layer was disrupted and detached by thorough scraping with a rubber policeman, and the fluid was drawn up for determination of cellular lithium and total protein. Lithium in the lysate was measured by atomic absorption spectrophotometry using a Perkin Elmer Model 2280 with a graphite furnace attachment. Standards were mixed using approximately the intracellular ionic content of the cell. Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Lithium uptake results were expressed as nanomoles of Li per milligram cell protein. In preliminary studies, lithium incorporation was measured at 12, 16, and 20 generations in culture with no differences found. All studies were done in cells in these early generations in culture. We believed that it would be helpful, in comparing the lithium incorporation data with data from other cell types, particularly the RBC, to calculate a lithium distribution ratio for the fibroblast. We showed that fibroblasts tend to be uniform in size with a volume
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of approximately 900 ixm3 (9.0 x 10-712,1)per cell by the use of the cell-sizing attachment of a Coulter/Counter; this value was independently confirmed by spinning down a large number of cells in medium with Co ~° EDTA added to serve as an extraeellular marker (Brading and Jones 1969). Using the l-hr lithium uptake for these cells (in nanomoles per milligram cell protein), the cell volume determined above (9 x 10-7p, l), an average determination for the protein content of a cell (2.5 x 10 4 p,g/cell), and an average concentration of Li in the supernatant (16 mM), we calculated the l-hr lithium distribution ratio or fibroblast "lithium ratio," as reported in Table 1. Comparison in vitro RBC lithium ratios were obtained for most subjects using the technique of Pandey and Davis (1977). Incorporation experiments, lithium ratio determinations, and subsequent calculation of results were performed with the investigator blind as to the identity of the subjects. All determinations reported were the results of averaging at least triplicate values for each sample. If experiments on a given subject were performed two or more times, results of each experiment were averaged to give the reported value. Statistical comparisons were made using Student's t-test and the Pearson-product moment correlation.
Table 1. In Vitro Lithium Ratios and Fibroblast Lithium Incorporation
Subject
Li level
Fibroblast l-hr Li incorp.
RBC in vitro Li ratio
(nmol/Li/p,g
Fibroblast I-hr Li
protein)
dist. ratio
code
Sex
Age
MST TSM EPH LPA SPA DIN HRO GDF SOK FFR
F M F F M F M F M F
49 40 39 55 40 27 51 48 40 68
0.7 0.2 1.0 0.8 0.9 1.0 0.8 0.8 0.2 0.2
0.41 0.36 0.48 O. 22 0.49 0.50 -0.34 -0.37
145.2 92.9 101.3 198.4 128.0 149.8 75.9 76.3 51.7 ! 19.3
2.53 1.62 1.76 3.45 2.23 2.61 1.32 1.33 0.90 2.08
45.7 11.2
0.7 0.3
0.40 0.09
113.9 43.4
1.98 0.76
66 43 26 35 28 32 46 41 51 50
----------
--0.46 0.35 0.39 0.33 0.32 0.41 0.28 0.58
97.3 115.8 69.0 72.4 111.4 75.7 70.2 89.8 90.0 157.7
1.69 2.01 1.20 1.26 1.94 1.32 1.22 1.56 1.57 2.74
41.8 12.2
---
0.39 O. 10
94.9 27.6
1.65 0.48
(mEq/liter)
Manic-depressives
M e a n SD
Controls CRF CRE BDS LIS
BDT SUL MKE HRD WSS BGR
Mean SD
M M F M F F M M F F
--
62
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R . E . B r e s l o w et
al.
Results Table 1 presents the data obtained from the 10 manic-depressive and 10 control subjects. These includes the sex, age, plasma lithium level, RBC in vitro lithium ratio, fibroblast l-hr lithium incorporation, and fibroblast 1-hr lithium distribution, or "fibroblast lithium ratio." Note in particular that all manic-depressives received lithium, while no control subjects received lithium. RBC data could not be obtained for two manic-depressives and two controls, so that RBC lithium ratio values are from the eight remaining subjects in each category. The lithium ratios derived from both cell systems were compared. For the control group, there was a high degree of correspondence in lithium distribution between the two cell types (r = 0.71, df = 6, p < 0.05, two-tailed test). This comparison was nonsignificant for the manic-depressive group (r = 0.30, df = 6, NS) and for both groups combined (r = 0.14, df = 14, NS). The mean RBC lithium ratio for eight manic-depressives was 0.40 _ 0.09, whereas eight controls had a mean ratio of 0.39 +_ 0.10. There were no significant differences between the two groups (t = 1.01, df = 14). In the fibroblasts of the control population, we found a mean of 94.9 _ 27.6 nmol lithium was incorporated per milligram of cell protein. This yielded a "lithium ratio" of 1.65 +_ 0.40 for the fibroblast, approximately four times higher than for RBCs. The fibroblasts of the manic-depressive population showed a mean lithium incorporation of 113.9 --+ 43.4 nmol of lithium per milligram cell protein, equivalent to a fibroblast lithium ratio of 1.98 _ 0.76. No significant difference was found between the two groups (t = 1.16, df = 18, NS). As can be seen from Figure 1, in both the RBC and the fibroblast systems, there is considerable overlap in the data. Many fibroblast metabolic and transport properties seem to vary with age. However, this sample showed no evidence of an age effect on fibroblast lithium incorporation (r = 0.13, NS) for the pooled sample of manic-depressives and controls. The values for each group considered separately were also not significant. The RBC lithium ratio also showed no relationship with age in the pooled group (r = - 0 . 3 2 , df = 14, NS) or when manic-depressives and controls were considered separately. In the manic-depressive population, both the fibroblast and RBC lithium ratios showed little correlation with plasma lithium level (r = 0.38 in both cases, NS), age of illness onset (r = 0.45 and - 0 . 6 2 , both NS), or duration of illness (r = - 0 . 4 9 and 0.08, respectively, both NS). - 200
.6-150
o
.5-
0•0
.4
._l--
°n o
_.~ - I00
mno I• I
.2=
-50
.I c
normol controls
monic depressives
~
25
.3
monic depressives
.c o o
normol controls
E e
Figure 1. Lithium ratios and lithium incorporation in erythrocytes and fibroblasts of manic-depressives and controls. (A) RBC in vitro Li ratio. (B) Cultured fibroblast in vitro Li ratio and l-hr Li incorporation. Li incorporation is shown in right-hand scale. (ll) male; (0) female.
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Discussion Our data support the possibility that the fibroblast is a valid model for the study of lithium transport mechanisms. The cultured fibroblasts have the same genetic material as the other somatic cells of the manic-depressive subjects from which they derive but have never been exposed to lithium. Therefore, they may provide a better source of comparison with the control populations, which also had ho lithium exposure. The data show a high correlation between the fibroblast lithium ratio and the in vitro RBC ratio in lithium-free control subjects. This finding further reinforces the possibility that the lithium extrusion mechanisms function in a similar fashion in both cell types within subjects. There is a higher equilibrium ratio in the fibroblast because of the lower electrical potential inside the cell. However, it still must be explained why there is such poor correlation between the fibroblast "lithium ratio" and the RBC lithium ratio in the manic-depressive group. This may be accounted for by the fact that chronic lithium treatment gradually changes the RBC lithium ratio by a specific inhibiting effect on the Na-Li countertransport system (Meltzer et al. 1977). No such effect would occur on the fibroblasts taken from manic-depressives, since the skin fibroblasts get little exposure to lithium and are grown in vitro for a number of generations without exposure to lithium. Because there is a factor altering the lithium ratio in one cell type (RBCs) but not the other (fibroblasts), the correlation between the two measures for this population is very low (r = 0.30). The fibroblast lithium ratio in the manic-depressive population is expected to be higher than in controls because of the hypothesized genetic defect in countertransport, as Ostrow et al. (1978) demonstrated in the RBC. With less efflux of lithium from the cell, because of the higher electrochemical gradient in the fibroblast, one would expect markedly accentuated differences in lithium distribution ratios between manic-depressives and controls in this system. The data do not support this. The mean fibroblast lithium ratio in the manic-depressive population was higher than in controls, but only by 20% (1.98 vs. 1.65), and the range was so great (0.90-3.45) that the overlap is considerable (see Fig. 1), yielding a nonsignificant difference. This finding could indicate either that there is a small difference in fibroblast lithium distribution ratios between the two populations but an insufficient number of subjects have been tested for it to show up or, what appears to be more likely, that there is no difference between the two populations on this measure. In either case, the current data do not support the hypothesis that an inherited membrane transport defect in countertransport exists generally in the somatic cells of manic-depressives. Previous studies in RBCs that demonstrated differences in lithium distribution between manic-depressives and controls were done using patients taking lithium, suggesting that the differences could be an artifact of the effect of chronic lithium treatment on the countertransport system. The present study establishes the technique of fibroblast cell culture as a new tool for the investigation of lithium transport differences in psychiatric populations. This method was employed in a comparison between a manic-depressive and a normal population, making it possible to circumvent the problem that manic-depressives are usually on chronic lithium treatment, which would distort the lithium transport parameters of their RBCs. Lithium responders were not compared with nonresponders, since all our patients were lithium responders, nor were unipolars and bipolars compared, since our population consisted entirely of bipolars and of a matched group of normals. It would be reasonable to assume, however, that if any differences were obtained they would be even greater
64
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R.E. Breslow et al.
between manic-depressives and normals than between responders and nonresponders or unipolars and bipolars. Therefore, it was particularly striking that the overlap was so great and no significant differences were found, leading to the conclusion that the lithium ratio is probably not a good measure for differentiating the two populations. This is not an unexpected result, as the current measure is a gross general measure of the overall equilibrium distribution inside and outside the cell. Further work is certainly indicated to look for more subtle differences in underlying transport mechanisms between the two populations. The use of specific inhibitors of transport pathways and special conditions of studying transport should make possible a much better delineation of the different pathways involved in establishing the lithium distribution ratio in the fibroblast. Perhaps the measurement of one of these pathways might reveal much greater differences in the two populations. This would enable us to study the etiology or pathogenesis of manic-depressive illness in terms of the specific membrane transport defect involved.
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Pandey GN, Davis JM (1979): A procedure for the determination of Li ratio in vitro of human erythrocytes. In Cooper TB, Gershon S (eds), Lithium: Controversies and Unresolved Issues, Amsterdam: Excerpta Medica, pp 969-975. Pandey GN, Dorus E, Davis JM, et al (1979a): Lithium transport in human red blood cells. Arch Gen Psychiatry 36:902-908. Pandey GN, Goel I, Davis, JM (1979b): Effect of neuroleptic drugs on lithium uptake by the human erythrocyte. Clin Pharmacol Ther 26:96-102. Ramsey TA, Frazer A, Mendels J, et al (1979): The erythrocyte lithium-plasma lithium ratio in patients with primary affective disorder. Arch Gen Psychiatry 36:457-461. Rybakowski J, Frazer A, Mendels J, et al (1978): Erythrocyte accumulation of the lithium ion in control subjects and patients with primary affective disorder. Commun Psychopharmacol 2:99-104. Rybakowski J, Frazer A, Mendels J, et al (1977): Prediction of the lithium ratio in man by means of an in vitro test. Clin Pharmacol Ther 22:465-469. Rybakowski J, Strzyzewski W (1976): Red blood cell lithium index and long term maintenance treatment. Lancet 1:1408-1409. Sacchetti E, Bottinelli S, Bellodi L, et ai (1977): Erythrocyte/plasma lithium ratios. Lancet 1:908. Schou M, Juel-Nielsen N, Stromgren E, et al (1954): The treatment of manic psychoses by the administration of lithium salts. J Neurol Neurosurg Psychiatry 17:250-260. Spitzer RL, Endicott J, Robins E (1981): Research Diagnostic Criteria for a Selected Group of Functional Disorders, 2rid ed. New York: New York Biometrics Research Division, New York State Psychiatric Institute. Szentistvanyi I, Janka Z (1979): Correlation between the lithium ratio and Na-dependent Li transport of red blood cells during lithium prophylaxis. Biol Psychiatry 14:973-977.