Lactose intolerance

1
Lactose intolerance
Antonio Cao
Mauro Congia
Dipartimento di Scienze Biomediche e
Biotecnologie Università di Cagliari ASL Nº8 Via
Jenner, 09121 Cagliari
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Curriculum Vitae Mauro Congia, M.D.
1) Dati Personali Data di nascita16,
Giugno 1957 Luogo di nascitaCagliari
2) Attuale Indirizzo Dipartimento di Scienze
Biomediche e Biotecnologie (DSBBT), Università di
Cagliari Via Jenner, 09121, Cagliari,
Italy Phone [phone] Fax
39-70-609-5558
3) Corso di studi -Laurea in medicina e chirurgia
nel 1982 con 110 e dichiarazione di
lode. -Specializzazione in Pediatria nel 1986 con
60/60 e dichiarazione di Lode. -Scuola di Sanità
Militare a Firenze nel Gennaio-Aprile 1985 e
Ufficiale Medico di Complemento Aprile
1985-Aprile 1986.
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Obbiettivi della presentazione

• Inquadramento nosologico delle varie condizioni
  associate alla ipolattasia

• Sintomatologia della intolleranza al lattosio,
  fattori che ne condizionano la variabilità e
  suggerimenti volti a ridurne la comparsa

• Meccanismo di controllo molecolare del promotore
  e ipotetico meccanismo di influenza sulla
  trascrizione dellmRNA del gene della lattasi che
  potrebbe essere alla base della ipolattasia

• Genetica della lattasi, implicazioni
  evoluzionistiche e prospettive future

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Lactose intolerance
Terminological differences and possible
confounding terms about lactose intolerance

• Hypolactasia (lactase nonpersistence, lactase
  restriction) means that there is low lactase
  activity in the jejunal mucosa

• Normolactasia (lactase persistence) means that
  there is persistent lactase activity comparable
  to the neonatal period

• Lactose maldigestion and lactose malabsorption
  are terms to describe a poor lactose hydrolysing
  capacity without symptoms

• Lactose intolerance should only be used for a
  clinical entity, describing symptomatic lactose
  maldigestion (20 of hypolactasic individuals)

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Hypolactasia
Lactase domain

• The gene is located in 2q21

Phlorizin hydrolase domain

• 17 Exons (49 Kb)

• 6 Kb mRNA

• 1927 Amino acids

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Hypolactasia
It is due to low levels of lactase-phlorizin
hydrolase (LPH) in the brush border of the
small-intestinal enterocytes.

• ?-glucosidase activity responsible for
  hydrolyzing phlorizin, a disaccharide found in
  roots and bark of plants of the family Rosaceae
  and some seaweeds

Lactase has two activities

• ?-galactosidase activity

?-1,4- galactosidic linkage
Lactase
Lactose
Galactose
Glucose
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Hypolactasia
Physiological factors involved in lactose
absorption

• LACTOSE DIGESTION

It is the rate-limiting step in the overall
process of absorption
Enzyme activity is greatest in the mid- jejunum,
decreasing both proximally and distally,
resulting in minimal activity in the proximal
duodenum and the terminal ileum

• Lactose is hydrolyzed

• Uptake of glucose and galactose is accomplished
  by the sodium-dependent glucose carrier

• Defects in this transporter result in severe
  diarrhea following carbohydrate intake

8
Hypolactasia
Lactase activity is high during infancy, when
milk is the main nutrient and in the vast
majority of humans decline with aging
The switch is genetically determined despite a
continued intake of lactose
9
Hypolactasia
Lactase deficiency may be primary or secondary
Three forms with primary deficiency of lactase
are recognized

• Congenital lactase deficiency
  (Rare)
• Developmental lactase deficiency (Prematurity)
• Adult lactase deficiency
  (Very common)

10
Hypolactasia
A forth condition of lactose intolerance having
serious consequences in infants but associated
with a normal presence of lactase in enterocytes
is the

• Congenital lactose intolerance

Abnormal absorption of lactose and other
disaccharides from the gastric mucosa
(lactosuria disappears when lactose is given
intraduodenally).
Vomiting, failure to thrive, dehydration, renal
tubular acidosis, aminoaciduria, liver damage,
lactosuria and cataracts.
A milk-free diet leads to rapid recovery, and
after 6 months of age a normal diet (with milk)
is well tolerated.
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Hypolactasia

• Congenital lactase deficiency

It is a rare recessive disease
Since 1966, 42 patients have been diagnosed in
Finland and 18 elsewhere.
Usually, the mother notes a watery diarrhea,
generally after the first feed of breast milk or
at the latest within the next 10 days.
Characterized by an almost total lack of LPH
activity in jejunal biopsy.
No mutation in the lactase gene (LCT) was found
in a Finnish patient with congenital lactase
deficiency.
12
Hypolactasia

• Developmental lactase deficiency

It results from low lactase levels and is a
consequence of prematurity
Lactase activity in the fetus increases late in
gestation 23rd week 10 of full-term between
25rd and 34rd is 30 of full-term between 34rd
and 35rd 70 of full-term
Premature infants born at 28 to 32 weeks
of gestation have reduced lactase
activity
13
Hypolactasia

• Adult lactase deficiency

It is the most common form of disaccharidase
deficiency
The prevalence of primary adult lactase
deficiency varies according to race
Of the worlds population, 75 is estimated to be
lactase-deficient
Race Persons of all races are affected, with
higher prevalence among Asian, African, and South
American persons.
Age of presentation variable (1-2 years among
Thai population to 1020 years among Finns)
Sex Males and females are affected equally.
14
Hypolactasia

• Symptoms after the ingestion of lactose

Lactose intolerance
Abdominal pain (crampy, often localized to the
periumbilical area or lower quadrant) Bloating
Flatulence Borborygmi may be audible to the
patient and on physical examination Diarrhea
(stools are usually bulky, frothy, and watery)
Vomiting (particularly in adolescents)

• Variability of symptoms

There is a high difference between patients in
the perception of symptoms
15
Hypolactasia

• Development of symptoms of lactose intolerance is
  related to several factors

1. Amount of lactose in the diet
2. Rate of gastric emptying

• If gastric emptying is symptoms
• Skim milk is more associated with symptoms
• Diarrhea following subtotal gastrectomy is often
  a result of lactose intolerance (gastric emptying
  is accelerated in patients with a
  gastrojejunostomy)

3. Small-intestinal transit time.

• More rapid small-intestinal transit makes
  symptoms more likely

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Hypolactasia

• Lab Studies

Lactose tolerance test
Measures serial blood glucose levels after oral
lactose load
sensitivity of 75 specificity of 96 The
diagnosis is confirmed if the serum glucose level
fails to increase by 20 g/dL above baseline
False-negative results in presence of diabetes
and small bowel bacterial overgrowth Abnormal
gastrointestinal emptying can also affect the
results
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Hypolactasia

• Lab Studies

Milk tolerance test
Administer 500 mL of milk and measure the blood
glucose level An increase of less than 9 mg/dL
indicates lactose malabsorption
Dietary elimination
Resolution of symptoms with elimination of
lactose-containing food products and resumption
of symptoms with the reintroduction are findings
suggestive of lactose intolerance.
Thus dietary elimination should be used only for
diagnosis of lactose intolerance
18
Hypolactasia

• Breath hydrogen test

This is the diagnostic test of choice
Subjects are administered lactose, after which
expired air samples are collected for 3 hours to
assess hydrogen gas concentrations
A rise in breath hydrogen concentration greater
than 20 parts per million over the baseline after
lactose ingestion suggests lactase deficiency
19
Hypolactasia

• Small bowel biopsy

This is the criterion standard It is invasive
and rarely performed Provides definitive
information The biopsy results may be normal if
deficiency is focal or patchy

• Genetic tests

They will be available soon. May be based on the
detection of C/T polymorphism at position -13910
upstream the LPH gene
20
Hypolactasia

• Secondary lactase deficiency

Treatment is directed at the underlying cause

• Morbidity

It is low and the condition is not lethal

• Possible complications

Osteopenia in lactase deficiency seems to be a
risk factor for osteoporosis (due to avoidance of
dairy products or interference of undigested
lactose with calcium absorption)
Consumption of milk in subjects with lactase
persistence has been associated with an increased
risk of cataract and of ovarian cancer (galactose
may be implicated)
21
Hypolactasia
Strategies that allow lactose maldigesters to
successfully incorporate dairy foods into their
diets
1. Consume small amounts of lactose-containing
foods
2. Chronic/repeated intake of lactose-containing
foods allows colonic bacteria to adapt and more
efficiently metabolize lactose
3. Co-ingest lactose-containing foods with a meal
4. Consider the form of the lactose-containing
food Hard cheeses, chocolate and higher fat
milks, and ice cream are well tolerated
5. Eat live culture yogurt
6. Utilize commercially available lactose
digestive aids
22
Hypolactasia

• Genetics AR

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Hypolactasia

• Genetics

Sequence and mapping of LPH in 1988 (Mantei et
al., Kruse et al.)
Fine mapping of LPH in 1993 (Harvey et al.)
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Hypolactasia

• Genetics and molecular defect transcriptional
  control

LPH gene
promoter
DNA
RNA
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Hypolactasia

• Genetics cooperative activation of the LPH
  promoter

• All mammals lactase genes examined so far contain
  binding sites for transcription factors Cdx2,
  HNF-1?, and GATA in their promoter

• A physical association between members of GATA-4
  or GATA-5 and HNF-1? results in the cooperative
  activation of the promoter of LPH

• The interaction
• occurs through the C-terminal zinc finger and
  basic regions of GATA-5 and the homeodomain of
  HNF-1?

G
G
H
TATAA
promoter
G
TATAA
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Hypolactasia

• Genetics

• An analysis of Finnish families identified a
  single base polymorphism 13.9 kb upstream of the
  human lactase gene that correlates with
  biochemically assayed lactase non-persistence and
  restricted the locus to a 47-kb interval on 2q21

• Two variants -13910 and -22018 kb upstream of the
  lactase gene, initially found in Finnish lactose
  intolerance families
• -13910 was also found in all French, U.S.,
  Italian, Korean and German and African cases

• The presence of the same DNA variants in
  non-persistence alleles in different, distantly
  related populations together suggests that the
  persistence variant is old, occurring long before
  the differentiation of these populations

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Hypolactasia

• Genetics

• the polymorphism could modify a transcription
  factor binding site (AP2), but the functional
  significance remains to be defined.

AP2 consensus sequence?
CCCCAGGC
C/C(-13910)
T/T(-13910)
28
Hypolactasia

• Genetics long-range cis-acting regulatory element

C/T 13910
G/A 22018
CCCCAGGC
AP2?
??
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Hypolactasia
Distribution of lactase phenotypes
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Hypolactasia
How to explain the high prevalence of lactase
persistence and its geographic distribution?
1) "calcium dependence hypothesis

• lactase persistence is high among Northern
  European populations. Indeed, rickets and
  osteomalacia were potent selective factors in the
  conditions of low solar irradiation
  characteristic of Northwestern Europe

2) milk dependence hypothesis

• lactase persistence is high among all nomadic
  populations of sub-Saharan area (Beja in Sudan,
  Tuaregs in Niger, Fulani in Nigeria, Tussi in
  Congo basin)

• pastoralists in highly arid environments maintain
  balance of water and electrolytes through
  plentiful milk supply

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Hypolactasia

• Genetics geographic distribution

All three hypotheses are supported by the
geographic distribution of high lactose digestion
capacity in adults, but are also confounded by
the shared ancestry of the population.
In 1997 Holden and Mace, using a comparative
method that takes the problem of phylogenetic
confounding into account suggested that lactase
persistence distribution is consequence of
adaptation to dairying.
The analysis does not support the hypothesis that
lactose digestion capacity is additionally
selected for either at high latitudes or in
highly arid environments

• Thus lactase persistence is an example of
  Gene-Culture co-evolution

Finally using maximum likelihood methods they
suggested that the evolution of milking preceded
the evolution of high lactose digestion.
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Hypolactasia
Future prospective oral gene therapy
Few hours after administration, there is
widespread and intense gene expression. Enzyme
activity persists up to six months after a single
application.
In a rat model, peroral application of
adeno-associated virus encoding ?-galactosidase
(to replace missing lactase)
can reverse lactose intolerance.
AAVlac after 3 days
AAVlac after 6 months
Control
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Hypolactasia
Future prospective Transgenic animals producing
low-lactose milk

• Transgenic animals carrying a hybrid gene in
  which the rat cDNA for LPH was placed downstream
  a murine mammary -specific promoter

• Female mice transgenic for this new gene not only
  produced lactase in the milk-producing cells, but
  also secreted it into the milk itself. Milk
  collected immediately on secretion had 50 less
  lactose than normal but if it was allowed to
  collect in the mammary gland, the level dropped
  even further to around 85 less lactose than
  usual

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Hypolactasia

• Conclusions

• Hypolactasia is the normal condition while
  persistence represents the variant

• There is no clear evidence for either conditions
  to predispose to other diseases

• People with hypolactasia generally can tolerate
  about 250 ml of milk without complains

• There are many commercially available milks and
  dairy products containing low amounts of lactose

• Lactase enzyme may be added to food containing
  lactose to increase digestibility of dairy
  products

• A DNA test for for the -13910 polymorphism
  associated with hypolactasia will be soon
  available

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Riferimenti Bibliografici
1.Jarvela I., Sabri Enattah N., Kokkonen J.,
Varilo T., Savilahti E. Peltonen L. (1998)
Assignment of the locus for congenital lactase
deficiency to 2q21, in the vicinity of but
separate from the lactase-phlorizin hydrolase
gene. Am J Hum Genet, 63, 1078. 2.Poggi V.,
Sebastio, G. (1991) Molecular analysis of the
lactase gene in the congenital lactase
deficiency. Am. J. Hum. Genet, 49 (suppl.),
105. 3.Swallow D.M., Poulter M. Hollox E.J.
(2001) Intolerance to lactose and other dietary
sugars. Drug Metab Dispos, 29, 513. 4.Enattah
N.S., Sahi T., Savilahti E., Terwilliger J.D.,
Peltonen L. Jarvela I. (2002) Identification of
a variant associated with adult-type
hypolactasia. Nat Genet, 30, 233.