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High performance liquid chromatography: principles andclinical applications

Ian M Bird

Detection of disease often relies on identifyingabnormal molecules or imbalanced ratios of normalmolecules in body fluids, these being reflections ofabnormal metabolism. The study of these moleculesand their interactions in both healthy and diseasedstates aids diagnosis and the monitoring of treatment.To carry out such investigations we need to identify,quantify, and ultimately purify the moleculesconcerned.One technique that is highly successful in achieving

all three objectives is column chromatography, andparticularly high performance liquid chromatography(HPLC).

General principles of column chromatographyThe underlying principle of chromatography is that

molecules not only dissolve in liquids but can also"dissolve" (absorb) on to or interact with the surface ofsolids. (Water will dissolve coffee, and coffee solutioncan also stain cloth.) If a molecule dissolved in a liquidis passed down a column of solid particles with which itinteracts it will move more slowly than the solvent,spending some time dissolved in the liquid (liquidphase) and some on the solid (solid phase). As suchmolecules take longer to pass down the column thanmolecules that do not interact with the solid phasethey emerge from the bottom of the column later(fig 1).The separation of molecules by chromatography

depends on exploiting the subtle differences in certainphysical properties of the molecules in samples: their

Department of solubility in water, solubility in organic solvents, netBiochemistry, University of positive or negative charge, and size. Separation ofEdinburgh, Edinburgh EH3 molecules according to these properties is described9YW respectively as adsorption (or normal phase), reverseIanM Bird, PHD, postdoctoral phase, ion exchange, or size exclusion chromatography.research fellow Each method requires a solid phase with different

Series edited by: surface properties (fig 2). For adsorption chromato-Drs Peter and John Hayes. graphy the solid phase must present- an aqueous

environment, whereas for reverse phase chromato-BrMedJ 1989;299:783-7 graphy it must present an organic environment; for ion

Columnof solidparticles(solid phase)

uLuuU UHA UULU uJ tUVPLFIG 1-Solvent ( l t ) containing sample molecules Afa and BRJ is continuously passed through a column.Solid material in the column can dissolve B on its surface, but not A, so that B spends part of its timedissolved on the solid surface, and moves more slowly down the column than A, which spends all ofits time inthe liquid

Normal phase chromatography

Solid phase Sample molecules

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FIG 2- Various forms of high performance liquid chromatography(HPLC) are based on solid phases of silica particles (0). In normalphase HPLC hydrophilic binding groups (-) on the silica surfaceattract hydrophilic (V) but not hydrophobic (-) molecules. In reversephase HPLC hydrophobic binding groups (- ) on the silica surfaceattract hydrophobic (*) but not hydrophilic (cp) molecules. In ionexchange HPLC thefunctional group attached may express a positive(-[1) or negative (---) charge and attract counter chargedmolecules (A , A '). In size exclusion HPLC the silica particles havebeen modified to create pores in the surface (@) to trap and retain small(0) but not large (0) molecules

exchange chromatography it must be charged, and forsize exclusion chromatography the solid phase must beable physically to retard smaller molecules as theymove through the column.Though the mechanism underlying separation will

be decided by the choice of solid phase medium, theactual separation achieved by normal phase, reversephase, and ion exchange chromatography will also beinfluenced by the choice of medium for the liquidphase, which competes with the solid phase for samplemolecules. Thus in normal phase chromatographysolvents of greater increasing polarity will moreeffectively remove polar molecules from the solidphase, and in reverse phase chromatography increas-ingly hydrophobic (organic) solvents will more readilyremove non-polar molecules from the solid phase.(This is equal but opposite to the normal phasemechanism-hence the term reverse phase.) In ionexchange chromatography the affinity of chargedmolecules for the solid phase is reduced when buffersof increasing salinity or changing pH, or both, are usedto suppress ionisation.

Often a liquid phase of changing composition is usedso that at the beginning of the method it is not as goodat dissolving the molecules of interest as is the solidphase and consequently the molecules spend almost alltheir time on the solid phase. As the composition of theliquid phase changes, however, it becomes a better

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solvent for the molecules of interest and so they areincreasingly removed from the solid phase into theliquid phase. This technique is called gradient elutionand can be applied to normal phase, reverse phase, and

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Retention time of sample moleculeFIG 3-Calculation ofefficiency ofa column from the equation n = 16(trYI(tw). The retention time ofthe peak (tr) and time takenfor the peakto emergefrom the column (tw) can be measuredfrom a chart recording

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FIG 4-( I up iwtng U ine nrgn VuaC prueSurOj il_r CUnsUolveniLS/iFLL Ualtverea ny a pUmp. AJ agradient elution is needed then two (or more) pumps are used to deliver the different solvent components to thecolumn through a mixing chamber. Samples are put in the flowing solvent by an injection valve. Moleculesemergingfrom the column are monitored continuously by an in line detector and the result recorded on a chartrecorder; (Bottom) A gradient HPLC system: HPLC column mounted on the side of a unit containingthe injection valve (extreme left); two HPLC pumps (left of centre, top and bottom); mixer (on bottompump); pump controller (centre bottom); ultraviolet detector (centre top); chart recorder (right) WATERSCHROMATOGRAPHY

ion exchange column chromatography but not to sizeexclusion chromatography, which depends only on thesize of the molecule.

High performance liquid chromatography columnsFor any chromatography column to be maximally

effective at retarding a sample molecule the columnmust be designed so that the sarple molecule hasample opportunity to come into contact with the solidphase. One means of achieving this is simply to makethe column bigger. Many columns may be a fewcentimetres long and have a volume < 10 ml, but somecolumns may be metres long and have a volume >1litre. The improvements achieved by altering columndimensions, are however, limited. A much moresuccessful approach is to make the particles of solidphase medium as small as possible. As the particle sizegets smaller (generally from 250 [tm to 5 im) thecolumn performance improves for two reasons: agreater area of solid phase is exposed to the samplemolecule (for any given column volume); and thevolume of solvent (and so diffusion time) between theparticles is reduced. Both these changes increase thechance that a sample molecule dissolved in the solventwill come into contact with, and so be retarded by, thesolid phase. But reducing the particle size of the solidphase brings practical problems. As the particle size isreduced and the space between the particles decreasesso the back pressure of the column increases. Thusmany solid phases (for example, those based oncellulose or organic resins) that have good physico-chemical properties at normal operating pressures(<0O5 kPa) can never give "high performance" withreduced particle size: they would collapse at the highworking pressures (up to 3- 5 kPa).

Fortunately, there are some solid phases which canwithstand these high operating pressures, and of thesethe most versatile and successful are those based onsilica. Particles of silica (5 [tm-10 iim) provide anexcellent solid phase for high performance normalphase chromatography. Chemical modification of thesurface provides materials which act as reverse phaseand ion exchange media. Alternatively, physicalmodification of silica to create tiny pores give the solidphase required for the retardation of smaller moleculesin size exclusion chromatography (fig 2). These basictypes of modified silica can be used to separate most, ifnot all, molecules of interest. Thus it is mainly columnswith solid phases derived from silica that form the basisof most high performance liquid chromatography(HPLC) methods.The best indication of how much better high perfor-

mance columns are than'normal columns is given bydetermining column efficiencies from the expression:

n= 16 (tr2)/(tW)where n is a measure of efficiency, t, is the retentiontime for a given peak of sample molecules, and tw is thewidth of the peak (fig 3). Typical values ofn for normalcolumns would be <500 but for high performancecolumns of only a few millilitres volume the value for ncan easily exceed 10000.

Equipment for high performance liquidchromatographyThe equipment used to operate HPLC columns can

be divided into two main areas, the delivery of solventand sample to the column and the detection ofmolecules emerging from the column. Figure 4 showsthe equipment used for solvent and sample delivery.Close regulation of the flow rate of solvent through thecolumn and of the composition of the solvent ingradient procedures is achieved by microprocessorfeedback systems controlling the pumps. Together

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with the use of a sample injection valve this makes anyHPLC method highly reproducible.To identify the appearance of the various molecules

being separated as they emerge from the column adetector is often attached "in line" with the columnoutlet. Usually detectors monitor the absorbance orfluorescence of the emerging molecules, but othermethods may sometimes be used. In line monitoringenables an immediate and continuous record of theseparation achieved to be recorded on paper by a chartrecorder.

Advantages and disadvantages ofHPLC as a routinemethodAn appropriate combination ofcolumn, solvent, and

detector in HPLC will allow the separation of one typeof molecule from others. Thus it can be used to rapidlyestablish a purification or assay method. In addition, itcan be used to identify and simultaneously quantifymany components of a complex mixture (such asmixtures of amino acids or steroids or drug meta-bolites), giving it a large advantage over radioimmuno-assay, or equivalent techniques, in which separateassays would be required. Furthermore, HPLC allowsunanticipated components in a sample to be detected,whereas in radioimmunoassay the assay is specific toonly one (or a few) molecules.HPLC does not lend itself readily to analysis of large

numbers of samples (unlike radioimmunoassay). Few

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methods of HPLC elution can be carried out in lessthan half an hour for one sample, but if samples arelimited in number a good throughput can be achievedwith automated systems of sample injection. Manysuch systems are now commercially available. Thoughthe initial cost for such automated apparatus may behigh, the running costs are often low. A furtherdisadvantage of HPLC is that, being a sensitiveanalytical technique, it is also sensitive to interferenceby sample or solvent contaminants and this may resultin artefacts or in an apparently successful methodbeing unusable in routine analysis. Using high puritysolvents and prepreparing samples appropriately willovercome these difficulties in most cases. Successfullyestablishing a routine HPLC method will often dependon such considerations.

Clinical applicationsHPLC has a considerable range of applications in

both clinical research and routine clinical analysis. Thefollowing examples illustrate the variety of moleculescommonly analysed by this method.

Measuring glycated haemoglobin, especially type lc,is a means of monitoring longer term plasma glucosecontrol in diabetic patients. As all the la, b, and c formsof glycated haemoglobin are less positively chargedthan normal haemoglobin at neutral pH, a method thatseparates proteins by charge should separate glycatedfrom normal haemoglobin. Methods based on chroma-

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FIG 5-Reverse phase HPLC separation ofplasma amino acids from normal and abnormal subjects after chemical modification ofamino acidswith orthophthalaldehyde. -(Courtesy ofG Blundell)

a. Amino acid profile obtainedfrom undiluted normal plasma. Peaks 3, 9, and 21 are not naturally occurring but were added as standardsb. 4mino acid profile obtainedfrom diluted (fourfold) plasmafrom a subject with maple syrup urine disease. Peaks 3, 9, and 21 were added as

standards. Note relatively high peaks 19, 23, and 24 (valine, isoleucine, and leucine) compared with other amino acidsI=aspartate, 2=glutamate, 3=homocysteic acid, 4=cysteine, 5=asparagine, 6=serine, 7=histidine, 8=glutamine, 9=homoserine, 10=

glycine, 11=threonine, 12=citrulline, 13=arginine, 14=alanine, 15=taurine, 16=tyrosine, 17=as-amino-n-butyrate, 18=ethanolamine, 19=valine, 20=methionine, 21=norvaline, 22=phenylalanine, 23=isoleucine, 24=leucine, 25=ornithine, 26=lysine



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tography from small cation exchange columns as wellas various electrophoresis methods have been usedsuccessfully to resolve glycated from non-glycatedhaemoglobins. A serious complication, however, isthat other forms of haemoglobin (such as HbF, HbS,and HbC) associated with haemoglobinopathiesmigrate with the glycated haemoglobins both in electro-phoresis and on cation exchange columns. Cationexchange HPLC, however, can resolve all the subtypesof glycated haemoglobin both from each other andfrom the F, S, and C forms. This form of assay showsexcellent precision with rapid separation, and manymanufacturers market HPLC systems dedicatedentirely to this purpose.The ability of HPLC to resolve closely related

molecules makes it the method of choice for detailedinvestigation of many congenital metabolic disordersor diseases. For instance, though amino acids inplasma or urine, or both, can be investigated withpaper chromatography, thin layer chromatography,or high voltage electrophoresis, these methods giverelatively poor separation and results are difficult toquantify. Ion exchange HPLC methods have beenmuch more successful in separating, identifying, andquantifying the main amino acid species in plasma andin urine. Chemical pretreatment of amino acid samplesto increase their solubility in organic media has enabled27 different amino acids to be resolved by reverse phaseHPLC (fig 5).2 The relative value of such methods isshown in maple syrup urine disease and phenyl-ketonuria. Maple syrup urine disease, which is causedby a deficiency of branched chain oxo-acid decarboxy-lases that results in an accumulation of leucine andoften of isoleucine and valine, can be detected by paperor thin layer chromatography or electrophoresis. Withraised concentrations of plasma phenylalanine,however, an amino acid profile is necesssary to find theunderlying cause. Phenylalanine concentration alonemay be raised, as in phenylketonuria, or may increaseas a consequence of hypertyrosinaemia or owing togeneralised hyperaminoacidaemia. A high resolutiontechnique like HPLC can differentiate between thesestates with the same basic method.

Other general types ofmolecule can also be separatedinto individual smaller molecules by HPLC to givemetabolic profile. With ion exchangeHPLC techniquesthe biogenic amines can be analysed. This is of value ininvestigating conditions such as phaeochromocytoma(associated with raised catecholamines) or carcinoidsyndrome (raised serotonin).

Abnormalities in purine or pyrimidine metabolismoften result in abnormalities in levels of nucleotides.Simmonds et al recently described an anion exchangeHPLC procedure for the separation of the majornucleotides and their corresponding deoxyderivatives.3Investigation of the erythrocyte nucleotide profiles ofsubjects showed that characteristic changes weredetectable in six out of eight purine or pyrimidinedisorders tested. The method can also be used on fetalblood and so allows antenatal screening for disorderssuch as adenosine deaminase deficiency (fig 6), whichleads to fetal immunodeficiency syndrome, as wellas more benign disorders such as xanthine oxidasedeficiency.

Reverse phase HPLC has been used successfullyfor identifying and quantifying individual urinaryporphyrins. Thus it is possible, for instance, to distin-guish between porphyria cutanea tarda (defectiveuroporphyrinogen decarboxylase), which resultsin highly carboxylated porphyrins, and porphyriavariegata (deficiency ofprotoporphyrin oxidase), whichresults in moderately carboxylated porphyrins.Many HPLC methods have been developed for the

study of vitamins and their metabolites. It is possible toseparate vitamin D from its 25-hydroxy and 1,25-

Blood from normal fetus

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Time (min) Time (min)FIG 6-Nucleotides and deoxvnucleotides from fetal erythrocvtehydrolvsates of normal and abnormal (adenosine deaminase deficient)subjects. Note increase in deoxvnucleotides dA TP and dADP in theabnormal subject. Separation wvas achieved bygradientanion exchangeHPLC. (Reproduiced with permission from Simmonds et a1!)

dihydroxy forms and so investigate the possibility thatreduced formation of the active dihydroxy form wasdue to impaired renal function or congenital abnor-malities.One of the main areas in which HPLC is used is in

therapeutic drug monitoring. Monitoring is beneficialunder a variety of circumstances-for example, whenthe therapeutic dose is close to the toxic dose, whensigns of toxicity are difficult to detect clinically, whenthe rate of metabolism varies widely between patients,or when drug metabolism is impaired owing to organdysfunction or altered by other drugs. Monitoringwhen rates of metabolism might vary is especiallyimportant if the drug metabolite is the therapeuticallyactive form or the toxic form.Drugs that are commonly monitored by HPLC

include antiasthmatic drugs (theophylline and itsactive metabolities, including caffeine), anticonvul-sants such as carbamazepine, phenobarbitone,phenytoin, ethosuximide, primidone and valproate,tricyclic antidepressants, and cardioactive drugs suchas procainamide and propranolol. In many cases thedrug may be monitored by radioimmunoassay, butwhen no antibody to the drug (or metabolite) exists, orif simultaneous measurement of a mixture of drugs ormetabolites is required then HPLC is more effective.4HPLC can readily be used to identify drug meta-

bolites that may be important in drug toxicity. This iswell illustrated by cyclosporin A, a powerful immuno-suppressant that has revolutionised organ transplan-tation, but which has potentially serious nephrotoxicside effects. The original radioimmunoassay methodshowed a correlation between concentrations ofcyclosporin A and nephrotoxicity that was not foundby HPLC. This difference was due to the lack ofspecificity of the antibody used: radioimmunoassaymeasured not only cyclosporin A but also several of itsmetabolites, whereas HPLC clearly separated cyclos-porin A from them. These differences imply that oneor more of the metabolites of cyclosporin A may beresponsible for nephrotoxicity, rather than cyclosporinA itself. Once the nephrotoxic metabolite has beenidentified by high resolution techniques such as HPLCthen a specific radioimmunoassay can be developed.HPLC is also widely used to identify causes of

poisoning. Common toxins that are freely availableinclude alcohols (ethanol, methanol, ethylene glycol),analgesics (aspirin, paracetamol), barbiturates, and

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GlossaryAdsorption (normal phase) HPLC-separation of molecules on the basis of theirsolubility in waterChiral molecules-those containing a carbon atom to which are bound four differentgroups. As the arrangement of these groups is asymmetric the molecule may exist in twoforms that are mirror images of each other yet have the same constituent partsGradient elution-a form of HPLC in which the composition of the liquid phase isaltered with time, so encouraging the transfer of molecules from the solid phase to theliquid phaseIon exchange HPLC-separation of molecules on the basis of their net charge

Liquid phase-the solvent passing through the HPLC columnReverse phase HPLC-separation of molecules on the basis of their solubility inorganic solventsSize exclusion HPLC-separation of molecules on the basis of their size

Solid phase-the surface of the solid particles (in an HPLC column) with whichmolecules can interact

paraquat, all of which can be rapidly identified byHPLC, as can those substances associated with drugabuse (opiates, amphetamines, hallucinogens, andcannabinoids).

HPLC and the futureThe practical limit to the efficiency of HPLC

columns is set by the solid phase. Improvements in theefficiency of silica based columns are unlikely, asreducing solid phase particle size to <5 Fm results inexcessively high back pressures, so improvementsneed new types of solid phases. Recently, highlyporous beads of organic resins to which functionalgroups can be attached have been successfully devel-oped. These columns give similar separations toconventional columns but at much lower back pres-sures. Columns formed from stacks of cellulose fibrediscs on to which functional groups are attached alsogive lower back pressures than conventional columns.The ability to pump columns at low pressure makes itpossible in many cases to speed up the analysis

by increasing flow rate. Alternatively, low pressurecolumns allow the use of pumps constructed of glassand plastic rather than steel and quartz, which is usefulwhen separating enzymes that may be inhibited byheavy metals or when the buffers used attack steel. Theincreasing availability of such columns may ultimatelyresult in the development of cheaper low pressureHPLC systems.

Alternative forms of silica based stationary phase arealso being developed. These have chiral functionalgroups attached that are capable of selectively bindingother chiral molecules. Many drugs are active inonly one of their two possible chiral forms, so thisdevelopment is of great importance clinically. Thesedevelopments and others yet to come will ensure a roleforHPLC in both research and routine clinical analysis.

ConclusionHPLC is an extremely powerful means of separating

molecules according to their solubility in water,solubility in organic solvents, net positive charge, netnegative charge, or size. The great variety of columnsand solvents available makes this method highlyadaptable to the investigation of a wide range ofmolecules, but the equipment is costly and throughputof samples is poor compared with other methods.When no other method is available or a complexmixture of components requires simultaneous assaythen the method is invaluable as a diagnostic tool and amonitor of treatment.

I thank Dr H A Simmonds and Dr G Blundell for providingthe illustrations as indicated and Mrs E Ward for her patienceand careful typing of the manuscript.

1 Goldstein DE, Little RR, Wiedmeyer H, England JD, McKenzie EM. Glycatedhemoglobin: methodologies and clinical applications. Clin Chem 1986;32:B64-70.

2 Blundell G, Brydon WG. High performance liquid chromatography of plasmaamino acids using orthophthalaldehyde derivatisation. Clin Chim Acta1987;170:79-84.

3 Simmods HA, Fairbanks LD, Mlorris GS, Webster DR, Harley EN. Alterederythrocyte nucleotide patterns are characteristic of inherited disorders ofpurine and pyrimidine metabolism. Clin Chim Acta 1988;171:197-210.

4 Matsumoto K, Kikuchi H, Kano S, Irn H, Takahashi H, Umino M. Automateddetermination of drugs in serum by liquid chromatography with columnswitching. I. Separation of anti-epileptic drugs and metabolites. Clin Chem1988;34:141-4.

BOOKS RECEIVED

Addiction

Stepping Out: Freedom from AlinorTranquillisers and Sleeping Pills. STrickett. (Pp 47; £1.50 paperback.)1988. Available from BroadcastingSupport Services, PO Box 7, LondonW3 6XJ.

OncologyCancer of the Bile Ducts and Pancreas.Ed P E Preece, A Cuschieri, R DRosin. (Pp xiii+328; figs; £52.)Philadelphia: Saunders, 1989. Distri-buted by Harcourt Brace Jovanovich.ISBN 0-7216-2631-9.Clinical Ocular Oncologv. D H Char.Illustrated b) J Weddell. (Pp x±402;figs; colour plates; £130.) New York:Churchill Livingstone, 1989. ISBN0-443-08569-2.

OphthalmologyDiagnosis and MAanagement of OrbitalTumors. J A Shields. (Pp xii +401; figs;£80.) Philadelphia: Saunders, 1989.Distributed by Harcourt Brace Jovan-ovich. ISBN 0-7216-2791-9.

Paediatrics

Caringfor Children with Chronic Illness:Issues and Strategies. R E K Stein. (Pp

xxx+301; $36.95.) New York:Springer, 1989. ISBN 0-8261-5900-1.Fighting, Teasing and Bullving: Simpleand E,J'fective Ways to Help Your Child.J Pearce. (Pp 96; figs; £1.99 paper-back.) Wellingborough: Thorsons,1989. ISBN 0-7225-1722-X.7'antrums and 7empers: 7ried-and-Tested Ways of Helping Y'our ChildCope with Strong Emotions. J Pearce.(Pp 96; figs; £1.99 paperback.)Wellingborough: Thorsons, 1989.ISBN 0-7225-1721-1.

Pharmacology

ABPI Data Sheet Compendium 1989-90 Together with the Code of'Practice jorthe Pharmaceutical Industriy. Associa-tion of the British PharmaceuticalIndustry. (Pp xvi+ 1854; £14.)London: Datapharm Publications,1989. ISBN 0-907102-00-X.South African Medicines Formulary. EdE A Conradie, J L Straughan. (Ppxvi+407; paperback, price not stated.)Pinelands: Medical Association ofSouth Africa, 1988. ISBN 0-86811-103-1.

Pharmacology - clinical

Drug Death: a Danger of Hospitaliza-tion. R P Hoffmann. (Pp xi+ 131;

$29.75.) Springfield: Thomas, 1989.ISBN 0-398-05554-8.MCQs in Clinical Pharmacology. BDavies, A Sinclair, S Jackson. (Pp219; £6.50 paperback.) Edinburgh:Churchill Livingstone, 1989. ISBN0-443-03729-9.

Physiology

Survival in Space: Medical Problems ofManned Spaceflight. R Harding. (Ppxix+227; figs; £14 95.) London:Routledge, 1989. ISBN 0-415-00253-2.

Psychiatry

Current Reviews in Psychiatry. No 3."Adolescent Psychiatry." P Hill.Series editors E S Paykel, H G Morgan.(Pp x+346; £29.95.) Edinburgh:Churchill Livingstone, 1989. ISBN0-443-02774-9.Self-Treatmentfor Phobias. K Sell. (Pp32; £2.25 each, or 10 copies for £20paperback.) 1989, Phobic Action,Greater London House, 547/551 HighRoad, London El1 4PR.

PsychologyAdvances in Suicidology. Vol 1. "Sui-cide and Its Prevention: the Role ofAttitude and Imitation." Ed R F W

Diekstra, R Maris, S Platt, ei al. (Ppx +371; $70 paperback.) Publishedunder the auspices of the World HealthOrganisation. Leiden: Brill, 1989.ISBN 90-04-08613-7.Against Therapy. Warning: Psychother-apy May be Hazardous to Your MentalHealth. J Masson. (Pp 320; £15.)London: Collins, 1989. ISBN 0-00-215092-1.Childhood and Human Nature: iheDevelopment of Personality. S Wolff.(Pp viii+242; £9.95 paperback.),London: Routledge, 1989. ISBN0-415-01129-9.Living with Tinnitus: Dealing with theRinging in Your Ears. R Hallam. (Pp128; figs; £4.99 paperback.) Welling-borough: Thorsons, 1989. ISBN0-7225-1801-3.

Longman Applied Psychology. "HealthPsychology." P G Harvey. Generaleditor P Feldman. (Pp x+137; figs;£4.95 paperback.) London: Longman,1989. ISBN 0-582-29623-4.

Rehabilitation

Everyday Aids and Appliances Articles-from the Brtilsh MedicalJournal. Ed GP Mulley. (Pp x+ll1; figs; £5.95,oserseas £7 paperback, including post-age, airmail overseas. BMA members'

£5.45, overseas £6.50 including post-age.) London British Medical _ournal,1989. In North America available fromthe American College of Physicians,PO Box 7777-R-0270, Philadelphia,PA 19175, USA. ISBN 0-7279-0241-5.Functional Electrical Stimulation.:Standing and WalkingAfterSpinal CordInjury. A R Kralj, T Bajd. (Pp vi + 198;figs; £66.50.) Florida: CRC Press,1989. Distributed by Wolfe MedicalPublications. ISBN 0-8493-4529-4.

Respiratory medicine

Asthma: Basic mechanisms and 7'hera-peutic Perspectives. Ed J R Vane, G AHiggs, S A Marsico, G Nistico. (Ppxiv+252; figs; £40.) Rome: PythagoraPress, 1989. ISBN 88-85852-01-7.

Statistics

Statistics with Cosnfidence: ConfidenceIntervals and Statistical Guidelines. EdM J Gardner, D Gi Altman. (Pp xviii+140; figs; £7.95, overseas £9.50 paper-back, including postage, airmail over-seas. BMA members £7.45, overseas£9 including postage.) London: BritishMedical J7ournal, 1989. In NorthAmerica available from the AmericanCollege of Physicians, PO Box 7777-R-0270, Philadelphia, PA 19175, USA.ISBN 0-7279-0222-9.



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