1243

REFERENCES

Chesney RW, Rosen JF, Hamstra AJ, DeLuca HF. Serum 1,25dihydroxyvitamin D levels in normal children and in vitamin D disorders. Am J Dis Child 1980; 134: 135-39. 13. Kruse K, Kracht U, Kruse U. Reference values for urinary calcium excretion and screening for hypercalciuria in children and adolescents. Eur J Pediatr 1984; 143: 25-31. 14. Farrington CJ, Chalmers AH. Gas-chromatographic estimation of urinary oxalate and its comparison with a colorimetric method. Clin

12.

1. Stickler GB, Beabout JW, Rigg BL. Clinical experience with 41 typical familial hypophosphatemic patients and 2 atypical nonfamilial cases. Mayo Clin Proc 1970; 45: 197-218. 2. Fraser D, Scriver CR. Familial forms of vitamin D-resistant rickets revisited. X-linked hypophosphatemia and autosomal recessive vitamin D dependency. Am J Clin Nutr 1976; 29: 1315-29. 3. Tieder M, Modai D, Samuel R, et al. Hereditary hypophosphatemic rickets with hypercalciuria. N Engl J Med 1985; 312: 611-17. 4. Isogna KL, Broadus AE, Gertner JM. Impaired phosphorus conservation and 1,25 dihydroxyvitamin D generation during phosphorus deprivation in familial hypophosphatemic rickets. J Clin Invest 1983; 71: 1562-69. 5. Lyles KW, Clark AG, Drezner MK. Serum 1,25-dihydroxyvitamin D levels in subjects with X-linked hypophosphatemic rickets and osteomalacia. Calcif Tissue Int 1982; 34: 125-30. 6. Glorieux FH, Scriver CR. Transport, metabolism and clinical use of inorganic phosphate in X-linked hypophosphataemia. In: Frame B, Parfitt AM, Duncan H, eds. Clinical aspects of metabolic bone disease. Amsterdam: Excerpta Medica, 1973: 421-23. 7. McEnery PT, Silverman FN, West CD. Acceleration of growth with combined vitamin D—phosphate therapy of hypophosphatemic resistant rickets. J Pediatr 1972; 80: 763-74. 8. Alon U, Brewer WH, Chan JCM. Nephrocalcinosis: detection by ultrasonography. Pediatrics 1983; 71: 970-73. 9. Goodyear PR, Kronick JB, Jequier S, Reade TM, Scriver CR. Nephrocalcinosis and its relationship to treatment of hereditary rickets. J Pediatr 1987; 111: 700-04. 10. Parvainen MT, Savolainen KE, Korhonen PH, Alhava EM, Visakorpi JK. An improved method for routine determination of vitamin D and its hydroxylated metabolites in serum from children and adults. Clin Chim Acta 1981; 114: 233-47. 11. Scharla S, Schmidt-Gayk H, Reichel H, Mayer E. A sensitive and simplified radioimmunoassay for 1,25-dihydroxivitamin D3. Clin Chim Acta 1984; 142: 325-28.

Chem 1979; 25: 1993-96. 15. De Santo NG, Di Iorio B, Capodicasa G, et al. Renal excretion of calcium, oxalate and magnesium between 3 and 13 years: the value of overnight urine. Contr Nephrol 1987; 58: 8-15. 16. Williams HE, Smith LH. Primary hyperoxaluria. In: Stanbury JB, Wyngarden JB, Fredricson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1983: 204-28. 17. Wadman SK, Sprang FJ, Heiden C, Ketting D. Quantitation or urinary phenylalanine metabolites in phenylketonuria. In: Bickel H, Hudson FP, Woolf LJ, eds. Phenylketonuria. Stuttgart: George Thieme Verlag, 1971: 65-72. 18. Brodehl J, Gellissen K, Weber H-P. Postnatal development of tubular phosphate reabsorption. Clin Nephrol 1982; 17: 163-71. 19. Robertson NG. Measurement of ionised calcium in biological fluids. Clin Chim Acta 1969; 24: 149-57. 20. Earnest DL, Johnson G, Williams HE, Admirand WH. Hyperoxaluria in patients with ileal resection: an abnormality in dietary oxalate absorption. Gastroenterology 1974; 66: 1114-22. 21. Malberti F, Surian M, Colussi G, Poggio F, Minoia C, Salvadeo A. Calcium carbonate: a suitable alternative to aluminium hydroxide as phosphate binder. Kidney Int 1988; 33 (suppl 24): S184-85. 22. Delvin EE, Glorieux FH. Serum 1,25-dihydroxyvitamin D concentration in hypophosphatemic vitamin D-resistant rickets. Calcif Tissue Int 1981; 33: 173-75. 23. Leumann EP. Primary hyperoxaluria: an important cause of renal failure in infancy. Int J Pediatr Nephrol 1985; 6: 13-16.

Paradoxical effect of bicarbonate

on

cytoplasmic pH

The effect of an abrupt rise in bicarbonate concentration on cytoplasmic pH was studied in human platelets suspended in a Tyrode’s buffer. Addition of bicarbonate raised extracellular pH but simultaneously caused pronounced cytoplasmic acidification. This effect may be due to combination of bicarbonate with hydrogen ions in extracellular fluid to form carbonic acid, which is converted by carbonic anhydrase to water and carbon dioxide. Bicarbonate ions do not diffuse rapidly across cell membranes, whereas carbon dioxide is highly diffusible and can combine with water in the cytoplasm, forming carbonic acid and reducing the intracellular pH. In accord with this acidification explanation by cytoplasmic bicarbonate was antagonised by acetazolamide (an inhibitor of carbonic anhydrase). Cytoplasmic acidification could contribute to adverse effects of intravenous sodium bicarbonate in patients with severe acidaemia. These findings add weight to the body of opinion that such treatment is both illogical and dangerous.

Introduction

importance of carbonic acid/bicarbonate as a physiological buffer has been appreciated for many years. The reversible hydration of carbon dioxide to carbonic acid and dissociation of this acid (to hydrogen and bicarbonate ions) are fundamental to acid-base physiology. The hydration and dehydration reactions are catalysed by carbonic anhydrase (carbonic dehydratase: EC 4.2.1.1). The Henderson-Hasselbach equation, derived by applying the The

law of mass action

to

these reactions,

states

that:

pH=pK+log [HCO,-]]

etP CO2 is the solubility of carbon dioxide, and P its partial pressure. Intravenous injection of sodium bicarbonate raises plasma pH and is advocated in treatment of life-threatening acidaemia.1,2 Nevertheless, because carbon dioxide can diffuse across cell membranes more rapidly than bicarbonate ions can, plasma pH is not directly related to where

oc

ADDRESS: Department of Clinical Pharmacology, United Medical and Dental Schools, Guy’s Hospital, London Bridge, London SE1 9RT, UK (Prof J. M. Ritter, FRCP, H. S. Doktor, BSc, N. Benjamin, MRCP). Correspondence to Prof J. M. Ritter.

1244

Fig 2-Effect of acetazolamide (01 mmoljl)

on

bicarbonate-

induced intracellular acidification.

resuspended platelets were transferred to a containing a magnetic stirrer and placed in a dualexcitation-wavelength fluorimeter (Deltascan, Photon Technology Inc, Richmond, Surrey, UK), at 22°C. Emitted light (530 nm) was measured during excitation with light of 500 nm and 440 nm alternately. When BCECF is excited at 500 nm the intensity of emitted light is strongly influenced by pH, whereas the intensity of emitted light is barely pH dependent with excitation at 440 nm.10 By expressing the signal as the ratio of the intensities at 500 nm and 440 nm it is therefore possible to correct, within limits, for dye concentration, cell density, and bleaching. Additions to the cuvette were made through an injection port directly into the stirred platelet 2-5 ml volumes of

cuvette

Fig 1-Effect of sodium bicarbonate (12 mmol/I) and sodium hydroxide (2 mmol/I) on intracellular pH of human platelets shown by fluorescent indicator (BCECF).

transcellular

fluid)3

or

pH (for example, within the cerebrospinal intracellular pH,4,s which determines the response to acid-base disorders.6 A high

to

metabolic bicarbonate ion concentration could therefore raise the extracellular pH while simultaneously reducing intracellular pH. In this study we have investigated the effect of an abrupt rise in extracellular bicarbonate concentration on intracellular pH, by means of human platelets loaded with a pH-sensitive fluorescent indicator.7

Methods were prepared on several occasions from each of ten non-smokers (six women aged 22-29 years, four drug-free healthy men aged 23-45 years). Venous blood was taken from an antecubital vein by means of a 19-gauge butterfly needle (Abbott, Sligo, Ireland), and prevented from clotting with trisodium citrate (final concentration 0-38%). Platelet-rich plasma was prepared from citrated blood by centrifugation at 750 g for 8 min at 20°C. The platelets were loaded with bis-carboxyethyl carboxy-fluorescein (BCECF) by incubation with its acetoxymethyl ester’ (BCECFAM : Calbiochem, Novabiochem, Nottingham, UK) 5 µmol/1, at 37°C for 30 min. The BCECF-loaded platelets were separated from free BCECF-AM by means of a column packed with ’Sepharose’ beads (CL-2B Pharmacia, Milton Keynes, UK) eluted with Tyrode’s solution (sodium chloride 140 mmol/1, glucose 5-6 mmol/1, Hepes 50 mmoljl, potassium chloride 2.8 mmol/1, KH2P04 0-8 mmol/1, magnesium chloride 0-84 mmol/1). All salts were of ’AnalaR’ grade from British Drug Houses (Poole, UK). The opaque fraction from the column containing BCECF-loaded platelets was collected. This fraction emerged several minutes before free BCECF-AM. Colforsin (Sigma, Poole, UK) 10 lunol/1 was added to the gel-filtered platelets and portions were spun at 1500 g for 10 min at 22°C. Supernatant was aspirated immediately and the platelet pellet resuspended briskly in 4 volumes of balanced salt solution per volume of gel-filtered platelets.

Platelets

suspension. In each experiment the platelet pellet from one portion of the suspension was resuspended in Tyrode’s solution in which we replaced sodium chloride with potassium chloride (125 mmol/1) so that we could obtain a calibration curve relating fluorescence ratio to pH by means of nigericin, an H+-K+ antiporter which sets [H+],/[H+L=[K+],/[K+] The pH in the cuvette was measured directly with a pH electrode (CE2: Philips Analytical, Cambridge, UK) after sequential additions of nigericin, sodium hydroxide, and hydrochloric acid. The resulting relation between pH and fluorescence ratio was analysed by linear regression; in all experiments the square of the correlation coefficient of this calibration curve was greater than 0-98. In most experiments, a platelet pellet was also resuspended in Tyrode’s solution containing physiological concentrations of sodium and chloride ions; in some experiments platelets were also resuspended in Tyrode’s solution in which sodium chloride was replaced by an equimolar concentration of chlorine chloride or of sodium or potassium aspartate, to study involvement of sodium and chloride ions. 5- (N,N-hexamethylene) amiloride (a potent inhibitor of Na+/H+ exchange;9 gift from Dr E. J. Cragoe, Lansdale, Pennsylvania, USA), dissolved in dimethyl sulphoxide was used to investigate further the effects of Na+/H+ exchange, and acetazolamide (Lederle, Gosport, UK; dissolved in water, diluted with an equal volume of dimethyl sulphoxide, and pH adjusted with hydrochloric acid) to investigate the effects of inhibiting carbonic anhydrase. 4,4’-acetamido-4’isothianocyanostilbene-2,2’-disulphonate (DIDS: Sigma) was used to investigate possible involvement of chloride/bicarbonate ion exchange.10 Results are expressed as mean and SEM; differences were evaluated by means of Student’s paired t test and were considered significant if p was below 0-05.

Results With the addition of sodium bicarbonate (12 mmol/1) to platelets suspended in Tyrode’s solution the intracellular pH fell rapidly (fig 1), whereas the extracellular pH measured directly with a pH electrode in the cuvette rose from 7-40 to 7-60. Sodium hydroxide (2 mmol/1) caused a similar rise in extracellular pH (to 7 61) but produced no

1245

alkaline value of extracellular pH caused by the bicarbonate

(fig 3). a fall in intracellular in platelets suspended in Tyrode’s solution in which sodium was replaced with choline chloride (fig 4); in sodium-free solution, addition of sodium chloride (40 mmol/1) after potassium bicarbonate caused partial recovery of intracellular pH. This effect of extracellular sodium was

Potassium bicarbonate also caused

pH

antagonised by 5-(N,N-hexamethylene)amiloride (added 120 s before sodium); there was more than 80% inhibition with 2 µmol/1 (n 6) and more than 95 % inhibition with 10 pmol/1 (n 6). There was no evidence of an effect of extracellular chloride or of chloride/bicarbonate exchange, on the intracellular acidification caused by bicarbonate; neither pretreatment for 60 min of platelets with DIDS (0-05 mmol/1), an inhibitor of both sodium-dependent and sodium-independent bicarbonate/chloride exchange," nor substitution of aspartate for chloride in the suspending medium affected the acidification significantly (n 6 in each case). =

=

Fig 3-Effect of bicarbonate (12 mmoljl) on cytoplasmic and extracellular pH in platelets in high-potassium Tyrode’s solution. Addition of

nigericin (0’01 mmol/1) raised intracellular pH

to

extracellular value

=

detectable change in intracellular pH (fig 1). The effect of sodium bicarbonate was quite consistent, causing cytoplasmic acidification of 0-24 (SEM 0-01) pH units (range 0-18-0-31 pH units) in 19 experiments (p < 0-001). In separate experiments (14 measurements on platelets from six subjects) platelet suspensions were acidified from pH 7 40 to pH 7 03 (002) units by addition of hydrochloric acid, to mimic an acidaemia in which therapeutic administration of bicarbonate might be considered. Subsequent addition of sodium bicarbonate (12 mmol/1) restored the extracellular pH to 7-44 (002) units, but caused cytoplasmic acidification of 0-24 (0-02) units (range 0-048-0-377 units). Acetazolamide almost completely prevented cytoplasmic acidification by bicarbonate in some preparations (fig 2) and in others it slowed but did not prevent the bicarbonateinduced fall in intracellular pH. Lower concentrations of acetazolamide caused less pronounced inhibition (data not shown). In platelets suspended in high-potassium Tyrode’s solution, addition of potassium bicarbonate (12 mmol/1) caused cytoplasmic acidification (fig 3). Under these conditions nigericin abolishes the extracellular/cytoplasmic pH gradient,8 and nigericin (0-01 mmol/1) added to platelets after potassium bicarbonate caused a pronounced rise in fluorescence ratio as the intracellular pH increased to the

Fig

4-Influence

of

sodium

on

bicarbonate-induced

cytoplasmic acidification in platelets in sodium-free (choline chloride 100 mmol/1) Tyrode’s solution. Potassium bicarbonate (12 mmoljl); sodium chloride (40 mmol/I).

Discussion In 1920

Jacobs showed that

a slightly alkaline carbon mixture is as toxic to tadpoles as a pure dioxide/bicarbonate carbon dioxide solution of the same concentration.4 He interpreted his findings as being due to the ability of carbon dioxide to enter cells freely, whereas bicarbonate cannot, carbonic acid dissociating in the cells to an extent determined by the conditions of equilibrium prevailing in the cytoplasm. He pointed out that the resulting intracellular hydrogen ion concentration could easily be higher than that of the surrounding medium. He obtained qualitative evidence supporting these ideas by means of the coloured flowers of Symphytum peregrinum, which contain a natural indicator, and which he exposed to solutions of various carbon dioxide and bicarbonate concentrations.5 Gesell and Hertzman3showed by means of a manganese dioxide electrode that bicarbonate can cause acidification of transcellular compartments; intravenous injection of sodium bicarbonate in dogs simultaneously increased the alkalinity of the circulating blood and the acidity of the cerebrospinal fluid. Sage and colleaguesll compared agonist-evoked changes in intracellular pH in human platelets in the presence and absence of physiological bicarbonate. They reported lower basal intracellular pH in the presence of bicarbonate than in its absence. Our findings accord with theirs and show the rapid effect on intracellular pH of an abrupt rise in bicarbonate concentration. The effect of acetazolamide implicates carbonic anhydrase in the rapid cytoplasmic acidification that results, consistent with carbonicanhydrase-catalysed dissociation of carbonic acid and differential diffusibility of bicarbonate and carbon dioxide.4 The experiments in sodium-free and chloride-free media and those with inhibitors of ion transport show that, in platelets, the effect of an acute rise in extracellular bicarbonate on intracellular pH is partly opposed by sodium/proton exchange but not by chloride/bicarbonate exchange. The relative importance of these and other transport mechanisms may well vary between different types of cell and in different conditions. 10,12 Carbonic anhydrase is ubiquitous.13 It is present in high concentrations within erythrocytes,14 but is also present on surface membranes of renal tubular cells 15 and of endothelial cells in culture.16 Our findings suggest that it is also present

1246

on the outer

surface of platelets. In any event, the presence of carbonic anhydrase on vascular cells as well as cells in the circulating blood provides a mechanism by which a high extracellular bicarbonate concentration could cause intracellular acidification in tissues with a rich capillary supply, such as the heart. This possibility is supported by the finding that intravenous sodium bicarbonate uniformly raises blood lactate concentration in patients with congestive heart failure, whereas sodium chloride does not.l’ Increased lactate production by cardiac and/or skeletal muscle could be due to intracellular acidification, as could the reduced myocardial oxygen consumption and negative inotropic effects observed.17,18 The usefulness of bicarbonate in lactic acidosis,19,20 diabetic ketoacidosis,21,22 and after cardiac arrest18,23 has been questioned. These reservations seem particularly important for therapy during cardiorespiratory arrest, when elimination of carbon dioxide by the lung is no longer under physiological control. However, many authorities continue to advocate intravenous sodium of treatment bicarbonate for life-threatening acidaemia,1,2,24,25 and the latest recommendations of the Resuscitation Council (UK) for prolonged resuscitation include "repeated doses of adrenaline and sodium bicarbonate" (50 mmol sodium bicarbonate intravenously every 5 min).26 Our study reemphasises that extracellular pH does not predict intracellular pH, and adds to the evidence that intravenous bicarbonate therapy for patients with severe acidaemia is both illogical and dangerous. We thank Prof 1. R. Cameron for invaluable advice.

REFERENCES 1. Andreoli TE. Disturbances in acid-base balance. In: Wyngaarden JB, Smith LH, eds. Cecil textbook of medicine, 18th ed. Philadelphia: WB Saunders, 1988: 549-58. 2. Cohen RD, Woods HF. Disturbances of acid-base homeostasis. In: Weatherall DJ, Ledingham JGG, Warrill DA, eds. Oxford textbook of medicine, 2nd ed. Oxford: Oxford University Press, 1987: 164-75. 3. Gesell R, Hertzman AB. The regulation of respiration. Am J Physiol 1926; 78: 610-29. 4. Jacobs MH. To what extent are the physiological effects of carbon dioxide due to hydrogen ions? Am J Physiol 1920; 51: 321-31. 5. Jacobs MH. The production of intracellular acidity by neutral and alkaline solutions containing carbon dioxide. Am J Physiol 1920; 53: 457-63. 6. Cameron IR. Acid-base disorders: analysis and treatment. Clin Endocrinol Metab 1980; 9: 529-41. 7. Rink TJ, Tsien RY, Pozzan T. Cytoplasmic pH and free Mg2+ in lymphocytes. J Cell Biol 1986; 95: 189-96. 8. Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 1979; 18: 2210-18. 9. Simchowitz L, Cragoe EJ Jr. Inhibition of chemotactic factor-activated Na+/H- exchange in human neutrophils by analogues of amiloride: structure-activity relationships in the amiloride series. Mol Pharmacol

1986; 30: 112-20. 10. Ganz MB, Boyarsky G, Sterzel B, Boron WF. Arginine vasopressin enhances pH regulation in the presence of HCO3- by stimulating three acid-base transport systems. Nature 1989; 337: 648-51. 11. Sage SO, Jobson TM, Rink TJ. Agonist-evoked changes in cytosolic pH and calcium concentration in human platelets: studies in physiological bicarbonate. J Physiol 1990; 420: 31-45. 12. Roos A, Boron WF. Intracellular pH. Physiol Rev 1981; 61: 296-434. 13. Knox WE. Enzyme patterns in fetal, adult and neoplasmic rat tissue. Basle: S. Karger, 1972. 14. Roughton FJW. Recent work on carbon dioxide transport by the blood. Physiol Rev 1943; 15: 241-96. 15. Maren TH, Ellison AC. A study of renal carbonic anhydrase. Mol Pharmacol 1967; 4: 503-08. 16. Ryan US, Whitney PL, Ryan JW. Localization of carbonic anhydrase on pulmonary artery endothelial cells in culture. J Appl Physiol 1983; 53: 914-19. 17. Bersin RM, Chatterjee K, Arieff AI. Metabolic and hemodynamic

18.

19.

20. 21. 22.

23. 24. 25.

consequences of sodium bicarbonate administration in patients with heart failure. Am J Med 1989; 87: 7-14. Ayus JC. Effect of bicarbonate administration on cardiac function. Am J Med 1989; 87: 5-6. Ryder REJ. The danger of high dose sodium bicarbonate m biguanideinduced lactic acidosis: the theory, the practice and alternative therapies. Br J Clin Pract 1987; 41: 730-37. Stackpool PW. Lactic acidosis: the case against bicarbonate therapy. Ann Intern Med 1986; 105: 276-79. Hale PJ, Crase J, Nattrass M. Metabolic effects of bicarbonate in the treatment of diabetic ketoacidosis. Br Med J 1984; 289: 1035-38. Morris RM, Murphy MB, Kitabchi AE. Bicarbonate therapy in severe diabetic ketoacidosis. Ann Intern Med 1986; 105: 836-40. Weil MH, Trevino RP, Rackow EC. Sodium bicarbonate during CPR. Does it help or hinder? Chest 1985; 88: 487. Robertson CE, Gourdie AL. Drugs in cardiopulmonary resuscitation. Prescribers’ J 1989; 29: 101-05. Wright AD. Diabetic emergencies in adults. Prescribers’ J 1989; 29:

147-54. 26. Chamberlain DA. Advanced life support. Revised recommendations of the Resuscitation Council (UK). Br Med J 1989; 299: 446-48.

BOOKSHELF Tomorrow’s Doctors: The Path to Successful Practice in the 1990s

Benjamin

H. Natelson. New York: Plenum. 1990.

Pp

288.

$19.95. ISBN 0-306431955. Medical practice is changing; the real professional world of doctors is not what it was when they entered medical school, and differs further from the private world of fantasy, speculation, and ignorance of medical school applicants. Benjamin Natelson has produced a survival guide for young doctors and medical students in the terra incognita of real medicine, for which medical school provided little preparation and no map. He stresses that medical school curricula overemphasise the "three Xs" (Dx, diagnosis; Px, prognosis; and Rx, treatment), producing "highly trained technicians", brimful of facts but unable to care for people. Implementing change is difficult: "Time, politics and a groundswell of student unrest will be required before the medical curriculum changes...", and since no single book can do that, he offers instead a guide to students and doctors on coping with the deficiencies of their training. Even if it is sometimes a little like Polonius’ avuncular speech to Hamlet, a surfeit of excellent advice become cloying, the book should nonetheless be commended to those who do not find the professional satisfaction they had expected in medicine. Natelson distinguishes physicians, concerned with the facts of medicine ("body plumbers") from doctors, who "put the facts into a human context that is meaningful to the patient". Physicians are characterised by the "three Bs", brash, boorish, and bullying, whereas the "three Cs", communication, caring, and creativity, convert physicians into complete doctors. The bulk of the book discusses "integrative medicine", arguing the importance of training for all medical practitioners in psychiatry, "physiatry" (rehabilitation medicine), and geriatrics, and considering such difficult patients as the "crocks". The principles acquired are then applied to the special problems of doctors themselves, "stress, money, sex and death". Natelson writes well (and would have succeeded in his alternative career of journalism), and the book is easy to read. Despite its strong American flavour, emphasising private practice, it contains much of relevance to future medicine everywhere. If it was read widely we might see less of the