Neurology India Medknow Publications on behalf of the Neurological Society of India ISSN: 0028-3886 EISSN: 1998-4022 Vol. 50, Num. 3, 2002, pp. 340-347

Neurology India, Vol. 50, No. 3, Sept, 2002, pp. 340-347

Case Report

Familial Hypodigoxinemic Membrane Na⁺-K⁺ ATPase Upregulatory Syndrome - Relation Between Digoxin Status and Cerebral Dominance

A.R. Kumar, P.A. Kurup*

Departments of Neurology and Biochemistry*, Medical College Hospital and University of Kerala*, Trivandrum, Kerala, India.
Correspondence to : Dr. P.A. Kurup, Gouri Sadan, T.C.4/1525, North of Cliff House, Kattu Road, Kowdiar, Trivandrum, Kerala, India.

Accepted for publication : 26th May, 2001.

Code Number: ni02097

Summary

A family with coexistence of hypotension, recurrent respiratory infection, motor tics, obsessive compulsive disorder, major depressive disorder, early onset osteoporosis, low body mass index, bulimia nervosa and healthy aging with longevity is described. The family members had hyposexual behavior, less tendency for spirituality, had no insomnia but a tendency towards increased somnolence, no addictive behaviour, had more bonding and affectionate behavior and were less creative with an average IQ. There was no vascular thrombosis, systemic neoplasm and neuronal degeneration in the index family. All members of the family were left hemispheric dominant. The level of serum digoxin, HMG CoA reductase activity and dolichol was found to be decreased in all with a corresponding increase in RBC Na⁺-K⁺ ATPase activity and serum ubiquinone magnesium level. There was increase in tyrosine catabolites and a reduction in tryptophan catabolites in serum. Total and individual glycosaminoglycan fractions, carbohydrate residues of glycoproteins, glycolipids, activity of GAG degrading enzymes and glycohydrolases were decreased in serum. The concentration of RBC membrane total GAG and carbohydrate residues of glycoproteins increased while cholesterol : phospholipid ratio of membrane decreased. The activity of free radical scavenging enzymes were increased while the concentration of free radicals decreased significantly. The same biochemical patterns were observed in left hemispheric dominance as opposed to right hemispheric dominance. The significance of these findings in the pathogenesis of these disorders is discussed.

Key words : Hypodigoxinemia, Dolichol, Ubiquinone, Membrane Na⁺-K⁺ ATPase, Obsessive compulsive disorder, Major depressive illness.

Introduction

Alteration in the endogenous membrane Na⁺-K⁺ ATPase inhibitor, digoxin, has been documented in depression as well as in essential hypertension.^1,2 Digoxin is a steroidal glycoside, synthesised in the human hypothalamus by the isoprenoid pathway and is also reported to modulate neuronal transmission.^3-5 The other isoprenoidal metabolites of significance are ubiquinone (regulate mitochondrial function), cholesterol (component of cellular membranes) and dolichol (regulate N-glycosylation of proteins). Therefore the isoprenoidal pathway, glycoconjugate metabolism, neuro-transmitter patterns, free radical metabolism and membrane composition were studied in the index family. The isoprenoid pathway was also compared in right hemispheric and left hemispheric dominance to find out whether hemispheric dominance plays a role in the genesis of the disorder.

Material and Methods

Three generations of the index family with coexistent hypotension, recurrent respiratory infection owing to immune deficiency, motor tics, obsessive compulsive disorder, major depressive disorder, early onset osteoporosis, low body mass index, bulimia nervosa and healthy aging were studied. OCD, major depressive disorder and bulimia nervosa were diagnosed by the DSM-IV criteria. Recurrent respiratory infections were diagnosed when there were more than 3 significant respiratory infections in a month that warranted treatment. Low body mass index was defined as a body mass index less than 18.5 kg/m². The family members were screened for behavioural patterns mentioned below; i) the criteria given in the handbook for the 16 personality factors questionnaire 16 PF was chosen after modification for defining spirituality, creativity and bonding / affection⁶ and ii) the criteria for insomnia and somnolence, sexual behaviour, addiction and eating behaviour were chosen from the DSM IV criteria. All the 15 alive affected members of the indexed family were chosen for the study except one family member - A¹, who died. Each patient also had an age and sex matched right handed/left hemispheric dominant control. In addition 15 normal left handed individuals who were right hemisphere dominant and 15 right handed individual who were left hemisphere dominant, between the age group 20-30 years, chosen by the dichotic listening test, were also studied for comparison of the same parameters. Informed consent was obtained from all the patients. All patients and control subjects were non-smokers (passive or active). The blood samples were collected from the patients before starting treatment. Activity of HMG CoA reductase of the plasma was determined using the method of Rao and Ramakrishnan by determing the ratio of HMG CoA to mevalonate.⁷ For the determination of the RBC Na⁺-K⁺ ATPase activity of the erythrocytre membrane, the procedure described by Wallach and Kamat was used.⁸ Digoxin in the plasma was determined using the procedure described by Arun et al.⁹ For estimation of ubiquinone and dolichol in the plasma, the procedure described by Palmer et al was used.¹⁰ Magnesium in the plasma was estimated by atomic absorption.¹¹ Tryptophan, tyrosine, serotonin and catecholamines were estimated by the procedures described in Methods of Biochemical analsysis.¹² Quinolinic acid content of plasma was estimated by HPLC. Morphine, strychnine and nicotine were estimated by the method described by Arun et al.¹³ Details of the procedures used for the estimation of total and individual GAG fractions, carbohydrate components of glycoproteins, activity of GAG degrading enzymes and glycohydrolases are described before.¹⁴ Serum glycolipids were estimated as described in Methods in Enzymology.⁸ Cholesterol was estimated by using commerical kits supplied by Sigma Chemicals, USA. Superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were estimated by the procedures described in Methods of enzymatic analysis.¹⁵ MDA (malondialdehyde), conjugated dienes, hydro-peroxides and reduced glutathione as well as iron binding capacity and ceruloplasmin were estimated by the procedures described in Methods of biochemical analysis.¹² Nitric oxide was estimated in the plasma by the method of Gabor and Allon.¹⁶Statistical analysis was done by students 't' test.

Results

Clinical features of the index family : The family tree of the indexed family is given in Fig.1 Motor tics was seen in 22% of the family members, OCD in 56%, major depressive disorder in 44%, hypotension in 72%, osteoporosis in 17%, low body mass index in 50%, recurrent respiratory infection with immunodeficiency in 44% and bulimia nervosa in 17%. The family members had hyposexual behaviour (80%), less tendency for spirituality and creativity (70%), had no insomnia but a tendency towards increased somnolence (60%), no addictive behaviour (80%) and had more bonding and affectionate behaviour (80%). One member of the family survived to 99 years of age and was healthy throughout his life span. There was total lack of incidence of vascular thrombosis, neuronal degeneration and systemic malignancies as well as healthy longevity (average life span of 85 years) in three generations of the indexed family. All members in the family were right handed - left hemispheric dominant (Table I).

Biochemical changes in the index family : The activity of HMG CoA reductase and the concentration of digoxin and dolichol were decreased in familial and left hemispheric dominant cases. The concentration of serum ubiquinone, the activity of erythrocyte membrane Na⁺-K⁺ ATPase and serum magnesium were increased. The opposite patterns were obtained in right hemispheric dominance (Table I). The concentration of serum tryptophan, quinolinic acid and serotonin was decreased while that of tyrosine, dopamine and noradrenaline was increased in the plasma of familial and left hemispheric dominant cases. Nicotine and strychnine were not detected in the plasma of familial and left hemispheric dominant cases. Morphine was detected in the plasma of familial cases (9.56ug/dL) and left hemispheric dominance (6.92ug/dL). The opposite patterns were obtained in right hemispheric dominance. Right hemispheric dominant individuals had no detectable morphine in the serum but had detected amounts of strychnine (9.52 ug/dL) and nicotine (2.07ug/dL) (Table II). The concentration of total glycosaminoglycans (GAG) and individual GAG fractions, carbohydrate components of glycoproteins and glycolipids decreased in the serum of familial cases. The activity of glycosaminoglycan (GAG) degrading enzymes and glycohydrolases was decreased in familial cases when compared to the controls. The concentration of total GAG and hexose and fucose residues of glycoproteins in the RBC membrane increased significantly in familial cases.

The concentration of RBC membrane cholesterol decreased while that of phospholipid increased resulting in a decreased cholesterol : phospholipid ratio (Table III). The activity of free radical scavenging enzymes, the concentration of reduced glutathione and ceruloplasmin and iron binding capacity increased significantly while the concentration of lipid peroxidation products and nitric oxide decreased significantly in familial cases.

Discussion

The decrease in the activity of HMG CoA reductase in familial cases suggests a downregulation of the isoprenoid pathway. There is a marked decrease in plasma digoxin and dolichol and this decrease may be a consequence of decreased channelling of intermediates of the isoprenoid pathway for their biosynthesis. The decrease in endogenous digoxin, a potent inhibitor of membrane Na⁺-K⁺ ATPase, can increase this enzyme activity. The stimulation of Na⁺- K⁺ ATPase by digoxin is known to cause an decrease in intracellular calcium and an increase in intracellular magnesium.¹⁷ Serum magnesium was assessed in familial cases and was found to be increased. Decrease in bone calcium load can lead to osteoporosis. The results showed that the concentration of tryptophan, quinolinic acid, serotonin, strychnine and nicotine was found to be lower in the plasma of patients with familial cases while that of tyrosine, dopamine, norepinephrine and morphine was higher. Nicotine and strychnine are synthesised from tryptophan and morphine from tyrosine.^13,18 Thus there is a decrease in tryptophan and its catabolites and increase in tyrosine and its catabolites in the patient's serum. This could be due to the fact digoxin can regulate neutral aminoacid transport system with a preferential promotion of tryptophan transport over tyrosine and that digoxin levels are low in familial cases.⁴ The increase in membrane Na⁺-K⁺ ATPase activity in familial cases could be due to the fact that the hyperpolarising neurotransmitters (dopamine, morphine and noradrenaline) are increased and the depolarising neuroactive compounds (serotonin, strychnine, nicotine and quinolinic acid are decreased. The low level of quinolinic acid, serotonin and strychnine can contribute to reduced excitatory glutamatergic transmission as they are all positive modulators of the NMDA receptor.¹⁹ In the presence of hypermagnesemia, the magnesium block on the NMDA receptor is strengthened leading on to reduced NMDA transmission.¹⁹ Reduced glutamatergic transmission can lead on to healthy aging and protect the brain from neuronal degeneration.¹⁹ The depressive syndrome noted in the family could be due to low serotonin. Decreased serotoninergic transmission has been related to depression. The presence of OCD syndrome in the family could also be related to serotonin depletion. Seratonin depletion has been related to obsessive psychopathology. The presence of motor tics could be related to increased dopaminergic transmission in the brain.²⁰ Deficiency of serotonin can lead to increased appetite and eating behaviour with bulimia in the family members.²⁰

Hypermagnesemia and decreased dolichol (required for N-glycosylation) levels can inhibit GAG, glycolipid and glycoprotein biosynthesis.²¹ The activity of GAG degrading enzymes and glycohydrolases decreased in the serum suggesting increased lysosomal stability. Intracellular hypermagnesemia also result in increased ubiquitin dependent proteolytic processing of glycoconjugates as it requires magnesium for its function. Defective lysosomal stability and defective degradation of glycoprotein - GAG complexes as in the case of tau protein/amyloid - HS proteoglycan complexes in Alzheimer's disease can lead on to brain aging.²² Membrane Na⁺-K⁺ ATPase stimulation could thus protect against neuronal aging and degeneration. A number of fucose and sialic acid containing natural ligands have been implicated in inflammatory responses and neoplastic transformation.²³ The decrease in fucose and sialic acid noted in these cases could lead on to an immunosuppressive state with recurrent respiratory infection and prevent malignant transformation. Decrease in bone structural glycosaminoglycans could contribute to osteoporosis. The downregulation of isoprenoid pathway can lead to decreased cholesterol synthesis and magnesium excess can stimulate phospholipid synthesis leading on to a decreased membrane cholesterol : phospholipid ratio. The concentration of total GAG and carbohydrate residues of glycoprotein increased in the RBC membrane and decreased in the serum suggesting their increased incorporation into the membrane. Hypermagnesemia can stimulate the activity of membrane trafficking enzymes - GTPases and lipid kinases. The change in membrane structure produced by alteration in glycoconjugates and cholesterol : phospholipid ratio can produce changes in the conformation of Na⁺-K⁺ ATPase resulting in further membrane Na⁺-K⁺ ATPase stimulation. The same changes can affect the lysosomal membrane increasing its stability.

The concentration of ubiquinone (free radical scavenger and component of mitochondrial electron transport chain) increased significantly in familial cases which may be the result of increased tyrosine levels, consequent to digoxin deficiency promoting tyrosine transport over tryptophan.⁴ The aromatic ring portion of ubiquinone is derived from tyrosine. The decrease in intracellular calcium can stabilise the mitochondrial PT pore and improve mitochondrial function.²⁴ Intracellular hypermagnesemia can lead on to increased ATP synthase activity. All this leads to improved efficiency of mitochondrial oxidative phosphorylation and reduced free radical generation. Decreased intracellular calcium also leads to decreased generation of NO by inhibiting the enzyme nitric oxide synthase and reduced peroxynitrite formation. The free radical scavenging enzyme activity, the concentration of antioxidants (ubiquinone, reduced glutathione, ceruloplasmin) and iron binding capacity increased significantly in familial cases suggesting increased free radical scavenging. The peroxisomal membrane is stabilised owing to membrane Na⁺-K⁺ ATPase stimulation related upregulation in membrane formation and leads to increased catalase activity. Hypermagnesemia leads to increased glutathione synthetase, glutathione peroxidase and glutathione reductase function. The stabilisation of the mitochondrial PT pore consequent to reduced intracellular calcium produces increased efficiency of superoxide dismutase activity. Mitochondrial dysfunction related free radical generation has been implicated in the pathogenesis of neuronal degeneration like PD, oncogenesis and inflammatory diseases. The reduced generation of free radicals leads to decreased incidence of neuronal degeneration and oncogenesis in the index family. Free radicals are required for lymphocyte activation and this leads to a hypoimmune response and increased respiratory infection owing to immunodeficiency. The decreased intracellular calcium and ceramide related stabilisation of the mitochondrial PT pore inhibits cytochrome C release and the caspase cascade.²⁴ Apoptosis has been implicated in neuronal degeneration and its inhibition protects against neuronal aging.

There is a decreased oncogenic tendency in the indexed family. Decreased intracellular calcium inactivates phospholipase C beta which results in decreased production of diacyglycerol (DAG) with resultant inactivation of protein kinase C and the MAP kinase cascade.²⁵ The intracellular hypermagnesemia can produce increase in the GTPase activity of the alpha-subunit of G protein resulting in ras-oncogene inactivation, as more of the ras is bound to GDP rather than GTP. Tumour suppressor gene, P₅₃ activation is increased owing to intracellular hypermagnesemia producing increased phosphorylation.²⁵ Decreased intracellular calcium inactivates the calcium dependent calcineurin signal transduction pathway involved in T cell activation and reduces the secretion of Interleukin 3, 4, 5, 6 and TNF alpha.²⁶ TNF alpha can also bring about apoptosis of the cell and this is inhibited. TNF alpha binds to its receptor TNFRI and activates the transcription factors NF-kB and AP-1 leading to induction of proinflammatory and immunomodulatory genes.²⁴ Low levels of TNF alpha can lead to immunosuppression in the family.

Hypermagnesemia can upregulate glucose transport as magnesium is required as a co-factor for cell membrane glucose transport. Intracellular hypermagnesemia can activate the phosphorylation reactions involved in protein tyrosine kinase receptor activity leading to increased insulin receptor activity. Intracellular hypermagnesemia can lead on to stimulation of glycolysis. Decrease in intracellular calcium can stabilise the mitochondrial PT pore and stimulate mitochondrial oxidative phosphorylation. Intracellular hypermagnesemia can also lead to a ATP synthase hyperactivity. This leads to increased glucose utilisation. Decrease in beta cell calcium and increase in magnesium can contribute to decreased insulin release from beta cells and hypoinsulinemia. Increased intracellular magnesium can produce hyperactivity of lipoprotein lipase producing increased catabolism of triglycerides rich lipoproteins and hypotriglyceridemia. In hypermagnesemia, Lecithin cholesterol acyl transferase (LCAT) is increased and there is increased formation of cholesterol esters in HDL. This results in increased HDL cholesterol. Magnesium excess has been reported to decrease LDL cholesterol levels also. Low insulin levels and increased triglyceride catabolism can be correlated with low body mass index noted in the family. Decreased in intracellular calcium can inactivate G-protein coupled angiotensin receptor producing hypotension and G protein coupled thrombin receptor and platelet activating factor producing decreased thrombosis observed in the family. Increased intracellular magnesium can lead to decreased thrombin and ADP/collagen induced platelet aggregation. Na⁺-K⁺ ATPase stimulation related decreased smooth muscle calcium and increased magnesium can contribute to vasodilatation and protect the family from ischaemia due to stroke and CAD. The family has a endogenous morphine excess syndrome. Morphine has been reported to have an effect on glucose metabolism.¹⁸ Intrathecal administration of morphine in the lumbar region causes a dose-dependent hypoglycemia. Morphine can also regulate insulin release from the beta cells with an inhibitory effect reported in some cases. Morphine has also got an immunosuppressive action. This could contribute to increased incidence of respiratory infections. Morphine excess can lead on to lack of addiction which has been noticed in the family membrane.¹⁸

The biochemical pattern obtained in the family correlated with the left hemispheric dominant state. In the left hemispheric dominant state there is a downregulated isoprenoid pathway, hypodigoxinemia, membrane Na⁺-K⁺ ATPase stimulation, decreased dolichol synthesis and elevated ubiquinone synthesis. There is an upregulated morphinergic, dopaminergic and noradrenergic transmission with a downregulated glutamatergic, cholinergic/nicotinic and serotoninergic transmission. There are no previous reports on a hypodigoxinemic syndrome in literature as also studies on biochemical differences between right and left hemispheric dominance.

References

1. Cowen PJ, Wood AJ : Biological markers of depression. Psychological Medicine 1991; 21 : 831-836.
2. Kramer HJ, Meyer Lehnert. H, Michel H et al : Endogenous natriuretics and ouabain like factors. Their roles in body fluid volume and B.P. regulation. Am J Hypertens 1991; 1 : 81-89.
3. Haupert GT : Sodium pump regulation by endogenous inhibition. Curr Top Membr Transport 1989; 34 : 345-348.
4. Hisaka A, Kasamatu S, Takenaga N : Absorption of a novel prodruig of DOPA. Drug-Metabol Dispos 1990; 18 : 621- 625.
5. Jyothi Augustine : Ph.D Thesis : University of Kerala Press, Trivandrum 1999.
6. Cattell RB, Eber HW, Tatsuoke MM : Handbook for the 16 PF : Institute of personality and ability testing publication, Illinois 1996.
7. Rao AV, Ramakrishnan S : Estimation of HMG CoA reductase activity. Clin Chem 1975; 21 : 1523-1528.
8. Colowick SP, Kaplan O : Methods in enzymology. Academic Press, New York. 1966.
9. Arun P, Ravi Kumar A, Leelamma S et al : Identification and estimation of endogenous digoxin in biological fluids and tissues by TLC and HPLC. Indian J Biochem Biophys 1998; 35 : 308-312.
10. Palmer DN, Maureen AA, Robert DJ : Separation of some neutral lipids by normal phase high performace liquid chromatography on a cyanopropyl column : ubiquinone, dolichol and cholesterol levels in sheep liver. Anal Biochem 1984; 140 : 315-319.
11. Price WJ : Spectrochemical analysis by atomic absorption : John Wiley Sons, New York. 1985.
12. Glick D : Methods of biochemical analysis : Inter Science, New York. 1971.
13. Arun P, Ravikumar A, Leelamma S et al : Endogenous alkaloids in the brain of rats loaded with tyrosine/tryptophan in the serum of patients of neurodegenerative and psychiatric disorders. Indian J Med Res 1998; 107 : 231- 238.
14. Manoj AJ, Kurup PA : Changes in the glycosaminoglycans and glycoproteins in the rat brain during protein calorie malnutrition. J Clin Biochem Nutr 1998; 25 : 149-157.
15. Bergmeyer HV : Methods of enzymatic analysis : Academic Press, New York .1978.
16. Gabor G, Allon N : Spectrofluorometric method for NO determination. Anal Biochem 1994; 220 : 16.
17. Haga H : Effects of dietary Mg2 ⁺ supplementation on diurnal variation of BP and plasma Na⁺-K⁺ ATPase activity in essential hypertension. Jpn Heart J 1992; 33 : 785-798.
18. Stefano GB, Scharrer B : Endogenous morphine and related opiates, a new class of chemical messengers. Advances in Neuroimmunology 1994; 4 : 57.
19. Greenamyre JT, Poter RHP : Anatomy and physiology of glutamate in CNS. Neurology 1994; 44 : 7-13. 20.
20. James R Gorman, Stevan Locke : Comprehensive text book of psychiatry : Williams and Wilkins, Baltimore 1989.
21. Jaya P, Kurup PA : Effect of magnesium deficiency on the metabolism of glycosaminoglycans in rats. J Bio Sci 1986; 10 : 487-497.
22. Beyreuther K, Masters CL : Alzheimer's disease-Tangle disentanglement. Nature 1996; 383 : 476-477.
23. Linstinsky JL, Siegal GP, Listinsky MC : Alpha-L-Fucose a potentially critical molecule in pathologic processes including neoplasia. Am J Clin Pathol 1998; 110 : 425-440.
24. Douglaus R Green, John C Reed : Mitochondria and apoptosis. Science 1998; 281 : 1309-1316.
25. Feinman R, Sawyer J, Hardin J et al : Cytogenetics and molecular genetics in multiple myeloma. Hematology- Oncology Clinics of North America 1997; 11 : 1-21.
26. Finkel TH : T-cell developoment and transmembrane signalling. Changing biologica responses through a unchanging receptor. Immunol Today 1991; 12 : 79-86.

Copyright 2002 - Neurology India. Also available online at http://www.neurologyindia.com

The following images related to this document are available:

Photo images