Gut 1994; 35: 1042-1046

Morphological method for the diagnosis ofhumanadult type hypolactasia

L Maiuri, M Rossi, V Raia, F Paparo, S Coletta, F Mazzeo, A Breglia, S Auricchio

AbstractThe primary adult type hypolactasia isthe most common form of geneticallydetermined disaccharidase deficiency.This study examined a large and homo-geneous population of the south of Italy:surgical biopsy specimens of proximaljejunum from 178 adult subjects have beenassayed for disaccharidase activities; theexpression of lactase protein and lactaseactivity has also been investigated ontissue sections by immunomorphologicaland enzymohistochemical techniques.Histograms of lactase to sucrase ratiowere found to provide a useful distribu-tion of the lactase activity; a lactase tosucrase ratio of 0 17 was found to showdiscrimination between tissues withpersistence of high lactase activity andtissues with adult type hypolactasia. In all28 subjects with persistent high lactaseactivity, a uniform distribution of lactaseprotein and lactase activity in all villusenterocytes was detected, whereas in all150 subjects with adult type hypolactasia avariable number of villus enterocytesfailed to express the lactase. Moreover inhypolactasic samples the lactase activityon tissue sections was constantly detectedlater than in samples with persistent highlactase activity. The absolute correlationbetween the immunohistochemical andenzymohistochemical features and theassessment of lactase activity in intestinalhomogenates suggests that the morpho-logical criteria are an alternative methodfor the diagnosis of adult type hypo-lactasia in human biopsy specimens fromproximal small jejunum.(Gut 1994; 35: 1042-1046)

Primary adult type hypolactasia is the mostcommon form of genetically determined dis-accharidase deficiency. In most humans and inmost mammals, intestinal lactase activitydeclines, on weaning, to about 5-10% of theactivity at birth. In a very high proportion,however, of the population in northernEurope, and many other milk drinking areas ofthe world, lactase activity persists into adultlife. These two phenotypes, namely lactasepersistent and lactase non-persistent (adulthypolactasia) are genetically determinedl 2 andare attributable to different alleles at oneautosomal locus. The effect of the alleleresponsible for the persistence of lactase afterchildhood is dominant to that of the allelecausing decline of lactase activity.3Accordingly, lactase deficiency in adults is

the recessive phenotype. By calculating thelactase/maltase or lactase/sucrase ratio3 4 insmall intestinal biopsy specimens, a trimodaldistribution was found, pointing to a homozy-gous lactase deficient phenotype, and hetero-zygous or homozygous lactase persistentphenotypes.5-9

Recently it has been shown by immuno-histochemical studies that in the proximaljejunum of subjects with persistent high lactaseactivity all the villus enterocytes express thelactase protein10 and lactase activity'1 on thebrush border whereas in adult hypolactasicsubjects the lactase protein'0 and lactaseactivity" were either absent or present on thebrush border of only some villus enterocytes.Surface immunohistochemical and enzymo-histochemical studies have shown that lactasepositive enterocytes are uniformly present onthe villus in the proximal small intestine ofsubjects with persistent high lactase activity,whereas in subjects with adult type hypo-lactasia scattered areas of enterocytes, whichexpress lactase protein and lactase activity arerandomly distributed on the villus wall. "I

In adult mammals the expression of lactaseprotein and lactase activity showed an un-expected degree of complexity in the pattern ofexpression along the villus and along theproximal-distal axis of the small intestine: inthe mid small intestine all enterocytes expressthe lactase protein'2 and lactase activity"Iwhereas in the most proximal and distal partsthe lactase is present only in patches of entero-cytes interrupted by enterocytes containing nodetectable protein12 or enzyme activity."IWe have studied a large white population

from the Campania region in the south of Italy.We carried out disaccharidase assays andimmunomorphological and enzymohisto-chemical analysis on the same specimens.Lactase persistent and lactase non-persistentphenotypes correlated closely with themorphological patterns. This suggested thepossibility of making the diagnosis of adulttype hypolactasia by morphological finding ofenterocytes not containing lactase protein orlactase activity, or both in the proximaljejunum.

Methods

HUMAN INTESTINAL SAMPLESProximal intestinal jejunal specimens weretaken from 178 adult patients who were receiv-ing intestinal resection. Informed consent wasobtained from all patients. All the subjectswere white, and were born in the south of Italy.They were aged from 22 to 78 years. The

Department ofPediatricsL MaiuriM RossiV RaiaS ColettaS Auricchio

and Department ofSurgery, University'Federico II' ofNaples,ItalyF Mazzeo

Departnent ofSurgery, OspedaleCardarelli, Naples,ItalyA Breglia

Correspondence to:Professor S Auricchio,Department of Pediatrics,University 'Federico H' ofNaples, via S Pansini 5,80131, Naples, Italy.Accepted for publication10 November 1993

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Morphological methodfor the diagnosis ofhuman adult type hypolactasia

patients needed surgery because of gastriccancer, duodenal or gastric ulcer, biliary tractdiseases, pancreatitis or gastric lymphoma.The patients were not given any cytostatic orantibiotic drugs - that is, neomicin, beforesurgery. The samples were taken about 5 cmdistal to the ligament ofTreitz and far from theintestinal lesion; in all subjects they were histo-logically normal. These specimens weredivided, half for histological studies and halffor enzyme assays.

ASSAY OF ENZYME ACTIVITIESSamples were homogenised at 2% concentra-tion in 2 mmol/I TRIS/HCl and 50 mmol/lmannitol (pH 7K1). Sucrase and maltase activi-ties were assayed according to the method ofAuricchio et al.13 The lactase activity wasassayed in the presence of p-chloromercuri-benzoate.14 For all samples with lowdisaccharidase activities, Zn(OH)2 deprotein-isation was used'5; this procedure eliminatesany interference with the glucose oxidaseassay, which is otherwise produced by the highconcentration of the intestinal homogenateneeded when determining low disaccharidaseactivities.'6 All chemical reagents wereobtained from Sigma Chemical Co, St Louis,MO, USA.

MONOCLONAL ANTIBODIES AGAINST HUMANLACTASEEight monoclonal antibodies against humanlactase were used in this study. The methodsand the characteristics of these antibodies,kindly donated by Dr Dallas Swallow(London) are reported in Green et al'7 and inour previous paper.'0

MONOCLONAL ANTIBODIES AGAINST HUMANSUCRASE AND HUMAN ISOMALTASEAntibodies CaCo 3/73 and HSI-9, kindly

. w ~~~4 4

Figure 1: Distribution of the lactase to sucrase ratio in the proximal small intestine of178 Neapolitan adults. Filled bars = subjects with persistent high lactase activity;hatched bars = hypolactasic subjects.

donated by Dr Quaroni were prepared aspreviously described.'8 19

HISTOLOGICAL STUDIES

ImmunohistochemistryOne part of the tissue was fixed in 4% formolacetate solution and embedded in paraffin waxaccording to routine histological procedure;another part was frozen immediately and keptat - 80°C until used. Five ,um sections wereincubated with mouse monoclonal antibodiesagainst human lactase, human sucrase, orhuman isomaltase, as previously described10(1:20-1:50, overnight at 4°C) followed bybiotinylated rabbit Ig directed against mouseIg (Dako, Copenhagen, Denmark) (1:200 forone hour) and the streptavidin peroxidasecomplex (Dako) (1:300 for one hour). Thesections were previously incubated with0-004% protease XXIV (Sigma) to enhancetheir immunoreactivity and also pretreatedwith 3% normal goat serum to prevent non-specific binding. Ten sections of eachspecimen were individually tested with all eightmonoclonal antilactase antibodies. Controlstaining reactions included replacement ofthe antilactase antibodies with (a) phosphatebuffer, (b) monoclonal antibody with anotherspecificity but with the same immunoglobulinisotype.

EnzymohistochemistryOne part of the tissue samples, immediatelyfrozen in liquid nitrogen, was cut with a coldmicrotome into 10 ,um thick sections. Thesections were transferred to slides at roomtemperature on which they thawed immedi-ately. They were then dried at room tempera-ture for 30 minutes. The experiments werecarried out according to Lojda and Kraml20 byusing 5Br-4Cl-3indolyl-,-Dfucoside (Sigma)as a substrate, which gives a blue reactionproduct.

In 10 tissues with persistent high lactaseactivity and in 35 hypolactasic samples, thelactase activity in villus attached enterocyteswere also measured in a quantitative wayaccording to Gutschmidt et al,21 as previouslydescribed.22

STATISTICAL METHODSThe distribution of each variable (lactase,sucrase, maltase, lactase/sucrase ratio) wasexamined by the EXAMINE procedure ofSPSS Pc + for the distribution.

Results

DISACCHARIDASE ACTIVITIESTraditionally subjects were classified as hypo-lactasic or lactase persistent by determining theratio of sucrase to lactase activity. A ratio ofabout 7-5 provided a convenient benchmark todiscriminate subjects with a genetic hypo-lactasia from those with persistent high lactase

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Maiuri, Rossi, Raia, Paparo, Coletta, Mazzeo, Breglia, Auricchio

Figure 2: Lactase immunoreactivity in the proximal small intestine of adult humans.(A) Expression of lactase protein in a subject with persistent high lactase activity.The lactase protein is uniformly distributed on the brush border of all villus enterocytes;the only interruptions of staining correspond to the goblet cells (arrow). (Monoclonalantibody mlac 1; paraffin wax embedded tissue, immunoperoxidase technique, originalmagnification x470); (B) Expression of lactase protein in a subject with adult typehypolactasia. The lactase protein is patchily distributed. Some villus enterocytes expresslactase protein (arrow) whereas the other enterocytes fail to show any immunoreactivit(arrow head). (Monoclonal antibody mlac 1; paraffin wax embedded tissue,immunoperoxidase technique, original magnification X470).

activity in necropsy samples of the lcjejunum8 and also in the surgical bi(samples of proximal jejunum in our S1population. IO

In our extended survey, however, wJcontains mainly hypolactasic subjects, rrof whom have very low lactase activihistograms of lactase/sucrase ratio are nuseful to discriminate the different groupsubjects (Kolgoroff-Smirnoff not significaTwenty eight subjects had persistent Ilactase activity with a lactase/sucrase r

>0 20, whereas 150 subjects had adult ihypolactasia with lactase/sucrase ratio <0therefore a lactase/sucrase ratio of 0 17 caiused to discriminate the two groups of subj(Fig 1).

In all subjects sucrase and maltase activwere within normal range.23

LIGHT MICROSCOPIC OBSERVATION

ImmunohistochemistryIn paraffin wax embedded or cryosisections from all subjects with persistent Ilactase activity, uniform and intense lacimmunoreactivity was seen at the brush boof villus enterocytes. Identical results ;obtained with all monoclonal antilacantibodies used in this study (Fig 2 (Intense staining of the brush bordersimilarly seen with the sucrase and isomalspecific antibodies.

In all 150 subjects with adult type h!lactasia enterocytes lacking lactase prcwere present. One hundred and thirty ninthese 150 subjects showed a patchy distrtion of immunoreactivity on the villi wit}the monoclonal antilactase antibodies.mosaic pattern was characterised by

presence of enterocytes with intensely stainedbrush border surrounded by mostly negativecells; enterocytes with faint staining of thebrush border were also seen (Fig 2 (B)). In 50of these 139 patients with a hypolactasia wecounted the number of positive cells: the per-centage of lactase positive enterocytes variedbetween 4% and 71X6% of total absorptivecells (mean (SD) 24-56 (12-4)). A significantcorrelation between overall lactase activity inthe mucosa and the percentage of enterocytesshowing staining of the brush border waspresent (r= 0 9) (Fig 3). In all the hypolactasicsamples in vitro biosynthetic studies haveshown reduced synthesis of the lactase protein,as previously reported.24 In contrast with theresults obtained with the anti-lactase anti-bodies, uniform and intense staining of thebrush border of all villus absorptive cells wasfound in the same subjects with sucrase andisomaltase specific antibodies.

Eleven of 150 subjects with adult typehypolactasia showed complete lack of stain-ing with all the antibodies to lactase with

the intense and homogeneous brush border stain-y ing with the anti-sucrase and anti-isomaltase

antibodies.

)wer ENZYMOHISTOCHEMISTRYcpsy In all subjects with persistent high lactasetudy activity the lactase activity was detected in all

villus enterocytes (Fig 4 (A)). By contrast, inhich all the subjects tested with adult type hypolac-iany tasia the lactase activity was patchily expressedties, (Fig 4 (B)) and it was present in the same villusnore enterocytes expressing the lactase protein.'s of In tissues with persistent high lactaseint). activity, the lactase activity is detected in allhigh villus enterocytes after only five minutes ofratio incubation with the substrate; by contrast, thetype lactase activity is still undetectable after 15'-14; minutes of incubation in hypolactasic samples;n be moreover, after 120 minutes of incubation thejects staining is much stronger in persistent than in

hypolactasic tissues (Fig 5), thus confirmingities our previous findings.22

DiscussionWe have examined 178 adult subjects bornin the Campania region around Naples in

tatic 80hightase g 60irder 60were -otase a 40

A)). ' X-was X 20 r= 087

5 10 15 20

Lactase specific activity (U/g protein)Figure 3: Correlation between lactase specific activity andpercentage of lactase containing enterocytes in smallintestinal biopsy specimens ofproximaljejunum from50 Neapolitan adults.

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Morphological methodfor the diagnosis of human adult type hypolactasia 1045

I h, *|1| ls 111

.1~~~~~~~~~~~~~~1

Figure 4: Expression and distribution of lactase activity by villus enterocytes ofproximalsmall intestine of adult humans. (A): Subject with persistent high lactase activity - anintense staining is evident on the brush border of all villus enterocytes. The crypt enterocytesare negative (enzymohistochemistry, original magnification x 100); (B): subject withadult type hypolactasia - the staining is evident on the brush border ofsome villusenterocytes; the other enterocytes are negative (enzymohistochemistry, originalmagnification xJI0).

southern Italy. The biopsy specimens wereobtained in the proximal small jejunum nearthe Treitz region.

In this extended survey, which containsmainly hypolactasic subjects, manv of whomhave very low lactase activities, thelactase/sucrase ratio was found to be veryuseful in discriminating between persistent andhypolactasic subjects. Using a lactase tosucrase ratio of 0d17 we found that 28 of 178Neapolitan adults had persistent high lactaseactivity, whereas 150 subjects had adult typehypolactasia. These results show that the adultpopulation in Naples has a low frequency ofpersistent high lactase activity, thus confirmingour previous findings.25

In all the subjects with persistent high lac-tase activity all the villus enterocytes expressthe lactase as shown by immunomorphologicalstaining with eight different monoclonal anti-lactase antibodies and by enzymocyto-chemistry; in all the hypolactasic subjects, incontrast, a variable number of villus entero-cytes failed to express the lactase. Thisabsolute correlation shows that morphologicalcriteria are an alternative method for the diag-nosis of adult type hypolactasia in humansbiopsy specimens from the proximal smalljejunum. It is not certain whether, in humans,as in the rabbit and rat, only lactase positive

> * Persistent high lactase activitycn

80 * Adult type hypolactasiaC: 80

70. 60

50c 0 -

40 r=09co 30En 20U,

0 r=09

0 15 30 60 90 120

Incubation time (mmn)Figure 5: Quantitative assessment of lactase activity invillus enterocytes of 10 subjects with persistent high lactaseactivity and 35 subjects with adult type hypolactasia.

enterocytes exist in the more distal parts of thesmall intestine. The diagnostic endoscopic orperoral biopsies are performed around theTreitz region, however, in the same regionwhere the tissue specimens used in thisstudy were taken from. The test proposedfor the diagnosis of adult type hypolactasiais performed on paraffin wax embedded orcryostatic samples, which are generally used aswell for other morphological purposes.Moreover, in hypolactasic tissues, the lactaseactivity is also still undetectable after 15minutes of incubation with the substrate,whereas in tissues with persistent high lactaseactivity it is detected after only five minutes ofincubation; therefore, the absence of stainingafter five minutes of incubation of frozen tissuesections with the substrate, is a sufficientcriterium to make diagnosis of adult typehypolactasia.

Only in a few hypolactasic subjects (11 of150 subjects) was the lactase absent from allvillus enterocytes; probably because a fewpositive enterocytes are present in closelyadjacent jejunal regions.Our findings are interesting not only as an

alternative diagnostic procedure, but also forunderstanding the mechanisms that underliethe adult type hypolactasia. The presence ofenterocytes without lactase activity is one ofthe mechanisms controlling the intestinallactase activity in the proximal small intestineof hypolactasic humans. The number of posi-tive enterocytes was in fact related to thelactase activity in these subjects. This is inagreement with the finding that most biopsyspecimens taken from the proximal jejunum ofhypolactasic adults show reduced synthesis oflactase protein in vitro and low levels of lactasemRNA.2628 Moreover, in hypolactasic tissuesthe quantitative enzymocytochemical assess-ment of lactase activity in villus attachedenterocytes shows that the enterocytes ofhypo-lactasic tissues also have less lactase activitythan enterocytes of persistent tissues.22 Thissuggests that in adult type hypolactasia thelactase protein is most probably present inreduced amounts or is less active.Mechanisms controlling lactase activity in

villus enterocytes in adult type hypolactasiaare being investigated in our laboratory by invitro biosynthetic studies and by in situhybridisation.

The authors thank Dr Dallas Swallow of the MRC, HumanBiochemical Genetics Unit, University College London, for thegift of the monoclonal anti-lactase antibodies and Dr LuigiGreco of the Department of Pediatrics, University 'Federico II'of Naples, Italy, for the statistical analysis of the data.This paper is dedicated to Professor Salvatore Auricchio on

the occasion of his 60th birthday.

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2 Semenza G, Auricchio S. Small intestinal disaccharidases.In: Scriver CR, Beaudet AL, Sly WS, Valle D, StanburyJB, Wyngaarden JB, et al, eds. The metabolic basis of inher-ited diseases. 6th ed. New York: McGraw-Hill, 1989:2975-97.

3 Ellestead-Sayed JJ, Haiworth JC, Hildes JA. Disaccharidaseconsumption and malabsorption in Canadian Indians.AmJyClin Nutr 1977; 30: 698-703.

4 Ellestead-Sayed JJ, Haiworth JC, Hildes JA. Disaccharidasemalabsorption and dietary patterns in two CanadianEskimo communities. Am J Clin Nutr 1978; 31: 1473-8.

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5 Flatz G, Rotthauve HW. The human lactase polymorphism:physiology and genetics of lactose absorption andmalabsorption. Prog Med Genet 1977; 2: 205-11.

6 Sahi T. The inheritance of selective adult-type lactosemalabsorption. Scand J Gastroenterol 1974; 9 (suppl 30):1-23.

7 Rosenzweig NS, Huang SS, Bayless TM. Transmission oflactose intolerance. Lancet 1967; ii: 777.

8 Ho MW, Povey S, Swallow D. Lactase polymorphism inBritish natives: estimating allele frequencies by enzymeassays in autopsy samples. Am J Hum Genet 1982; 34:650-7.

9 Flatz G. Gene dosage effect of intestinal lactase activitydemonstrated in vivo. AmJ Hum Genet 1984; 36: 306-10.

10 Maiuri L, Raia V, Potter J, Swallow D, Ho MW, Fiocca R,et al. Mosaic pattern of lactase expression by villus entero-cytes in human adult-type hypolactasia. Gastroenterology1991; 100: 359-69.

11 Maiuri L, Rossi M, Raia V, Paparo F, Garipoli V, AuricchioS. Surface staining on the villus of lactase protein and lac-tase activity in adult-type hypolactasia. Gastroenterology1993; 105: 708-14.

12 Maiuri L, Rossi M, Raia V, D'Auria S, Swallow D, QuaroniA, et al. Patchy expression of lactase protein in adultrabbit and rat intestine. Gastroenterology 1992; 103:1739-46.

13 Auricchio S, Rubino A, Tosi R, Semenza G, Kistler H,Prader A. Disaccharidase activities in human intestinalmucosa. Enzymol Biol Clin 1963; 3: 192-208.

14 Asp NG, Dahlqvist A. Human small intestinal B-galacto-sidases. Specific assay of three different enzymes.Anal Biochem 1972; 47: 527-38.

15 Sebastio G, Huzinker W, Ballabio A, Auricchio S, SemenzaG. On the primary site of control in the spontaneousdevelopment of small intestinal sucrase-isomaltase afterbirth. FEBS Lett 1986; 208: 460-4.

16 Ciccimarra F, Starace E, Vegnente A. Osservazioni sulmetodo di dosaggio delle attivita' disaccaridasiche nellamucosa intestinale umana. Boll Soc Ital Bio Sper 1968; 45:336-9.

17 Green FR, Greenwell P, Dickson L, Griffiths B, Noades J,Swallow DM. Expression of the ABH, Lewis, and relatedantigens on the glycoproteins of the human jejunal brushborder. In: J R Harris, ed. Immunological aspects. London:Plenum, 1988; 12: 119-45.

18 Quaroni A. Crypt cell antigen expression in human tumorcolonic cell lines. Analysis with a panel of monoclonal

antibodies to Caco-2 luminal membrane components._JNatl Cancer Inst 1986; 76: 571-85.

19 Beaulieu JF, Nichols B, Quaroni A. Post-translationalregulation of sucrase-isomaltase expression in intes-tinal crypt and villus cells. J Biol Chem 1989; 264:20000-11.

20 Lojda Z, Kraml J. Indigogenic methods for glycosidases III.An improved method with 4-Cl-5-Br-3-Indolyl-3-D-Fucoside and its application in studies of enzymes in theintestine, kidney and other tissues. Histochemie 1971; 25:195-207.

21 Gutschmidt S, Emde C. Early changes in brush borderdisaccharidase kinetics in rat jejunum following sub-cutaneous administration of tetraiodothyronine.Histochemistry 1981; 71: 189-98.

22 Maiuri L, Rossi M, Raia V, Paparo F, Auricchio S. Lactaseactivity of the enterocytes of human proximal jejunumwith adult-type hypolactasia. In: Auricchio S, Semenza G,eds. Commonfood intolerances 2:milk in human nutrition andadult-type hypolaciasia. Basle: Dyn Nutr Res 3, Karger,1993: 161-7.

23 Haemmerli U, Kistler HJ, Hamman T, Marthaler T,Semenza G, Auricchio S. Acquired milk intolerance in theadult caused by lactose malabsorption due to a selectivedeficiency of intestinal lactase activity. Am J Med 1965;38: 7-15.

24 Rossi M, Maiuri L, Fusco MI, Danielsen EM, Auricchio S.The human adult-type hypolactasia is a heterogeneouscondition in in vitro biosynthetic studies. In: Auricchio S,Semenza G, eds. Common food intolerances 2: milk inhuman nutntion and adult-type hypolactasia. Basle: DynNutr Res 3, Karger, 1993: 174-87.

25 Rinaldi E, Albini L, Costagliola C, De Rosa G, AuricchioG, De Vizia B, et al. High frequency of lactose absorbersamong adults with idiopathic senile and presenile cataractin a population with a high prevalence of primary adultlactose malabsorption. Lancet 1984; i: 355-7.

26 Sebastio G, Villa M, Sartorio R, Guzzetta V, Poggi V,Auricchio S, et al. The control of lactase in humanadult-type hypolactasia and in weaning rabbits and rats.Am3JHum Genet 1989; 45: 489-97.

27 Witte JM, Lloyd M, Lorenzson V, Korsmio H, Olsen W.The biosynthetic basis of adult lactase deficiency.J Clin Invest 1990; 86: 1338-42.

28 Lloyd M, Mevissen G, Fisher M, Olsen W, Goodspeed D,Genini M, et al. Regulation of intestinal lactase in adulthypolactasia. J Clin Invest 1992; 89: 524-9.

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