Basic Science
Gastrointestinal physiology
John McLaughlin
Abstract
This contribution focuses on the gastrointestinal tract and its ability
to absorb nutrients, water and electrolytes, and also how it forms an
­effective barrier against potentially harmful contents, such as bacteria.
Its structure and function are also discussed.
Keywords gastrointestinal; physiology
The gastrointestinal tract must not only absorb nutrients, water
and electrolytes, but must also form an effective barrier against
the ingress of potentially harmful contents, such as bacteria. Its
structure and function are highly adapted to serve these conflict-
ing roles.
Secretion of gastric acid
The primary reason for secreting gastric acid is to kill ingested
microorganisms. This appears less important in the developed
world and acid secretion is pharmacologically stopped with
impunity in millions of individuals. Acid denatures proteins, but
gastric enzymes and defence molecules are pH-adapted to allow
digestion to begin. At a pH of about 1 after a meal, gastric acid
is injurious to tissues except the highly adapted gastric mucosa.
A gel of mucus coats the epithelium, and bicarbonate is secreted
locally so that the pH adjacent to the cell surface is 6–7. A surface
coating of mucus also provides defence against autoproteolysis,
serving as a gel with a progressive pH gradient occurring from
the cell surface to the lumen. The epithelium is further protected
by a variety of factors including:
• prostaglandins (PGE2 in particular; its synthesis is blocked by
non-steroidal anti-inflammatory drugs, majorly contributing
to their ulcerogenicity)
• epidermal growth factors (e.g. heparin-binding epidermal
growth factors, amphiregulin)
• ‘trefoil’ peptides which are secreted into the lumen and may
monitor for damage and protect the mucosa.
Hydrochloric acid is secreted by parietal cells in the gastric
body (oxyntic mucosa), which express the H+–K+ ATPase or
proton pump. When stimulated, particularly by histamine, ­proton
John McLaughlin FRCP is a Senior Lecturer in Medicine at Manchester
University, Manchester and Honorary Consultant in Gastroenterology
at Hope Hospital, Salford, UK. He is Clinical Director of the
Gastrointestinal Physiology service. Conflicts of interest: none
declared.
SURGERY 27:6
pumps are rapidly recruited to the apical surface by fusion of
a vast intracellular canalicular membrane network and actively
extrude H+ into the lumen against a concentration gradient of
106 (the largest concentration gradient in human physiology). H+
derives from the action of the enzyme carbonic anhydrase, which
is abundant in parietal cells (CO2+H2O→H2CO3→HCO3−+H+).
Chloride is secreted in parallel via cyclic AMP-dependent apical
channels.
Control secretion of gastric acid is intrinsic and extrinsic, and
occurs in three phases.
The cephalic phase accounts for about 40% of total acid secre-
tion and is triggered by food in the mouth, although the sight,
smell or thought of food can trigger this, as can any conditioned
reflex (Pavlov’s dogs secreted acid in response to a mealtime bell
when food was not given). It is a vagal mechanism and is virtu-
ally abolished by vagotomy. This is the rationale for vagotomy
in the historical management of acid peptic disease, particularly
ulcers. It is mediated by post-ganglionic cholinergic fibres acting
on muscarinic (M3) receptors on the parietal cell.
The gastric phase is triggered by food in the stomach, particu-
larly l-aromatic amino acids (l-tryptophan, l-phenylalanine) and
small peptides liberated from initial digestion of protein, which
directly stimulate the release of the hormone gastrin from antral
G-cells. The sensory mechanism is not confirmed, but recent evi-
dence suggests that the extracellular calcium receptor (originally
cloned from parathyroid cells) acts as a polymodal nutrient sen-
sor expressed by G-cells. Mechanical stretch also has a role via
intrinsic neural reflexes and the vagal efferent nerves produce a
gastrin-releasing peptide. Alcohol and caffeine further stimulate
acid secretion.
Intestinal phase – food entering the intestine stimulates about
10% of acid secretion, which will persist with purely post-pyloric
tube feeding. G-cells are also present in the duodenum, predomi-
nantly secreting gastrin-28 which has a longer circulating half-
life than gastrin-14, the predominant antral G-cell product (see
below). The intestinal phase is more complex because inhibi-
tory hormones are also released, particularly in response to fat
(cholecystokinin (CCK), peptide YY) and acid (secretin, gastric
inhibitory polypeptide). These inhibitory effects constitute the
so-called ‘enterogastrone’ mechanism, and also contribute to
slowing gastric emptying, particularly after fatty meals. The acid
hypersecretion and hypergastrinaemia in surgical short bowel
probably reflects the functional loss of enterogastrones because
their tissue source has been removed surgically.
Gastrin and the feedback control of secretion of gastric acid:
gastrin is a regulatory peptide but is not a major direct regulator
of acid secretion by parietal cells. Amidated gastrins, the active
moiety at the CCK-2 (CCK, gastrin) receptor, are produced by
cleavage and post-translational modification from the prepro-
gastrin precursor, the initial translational product of the gastrin
gene. There is increasing evidence the progastrin has biological
activity, related to cell proliferation and differentiation. The main
target is the gastrin/CCK-2 receptor on the histamine-secret-
ing enterochromaffin-like cell, not the parietal cell as had been
thought. Histamine is secreted to act in a paracrine manner on
nearby parietal cells, operating at H2-receptors to stimulate acid
secretion via the mechanisms discussed above. Gastrin is trophic
225 © 2009 Published by Elsevier Ltd.
 Basic Science
to the oxyntic mucosa indirectly via epidermal growth factors,
which leads to the thickened folds found in Zollinger–Ellison
syndrome. The enterochromaffin-like cell also operates under
vagus nerve control, probably via pituitary adenylate cyclase-
activating peptide.
There is also an epithelial inhibitory mechanism in which a fall
in pH leads to an increase in the secretion of somatostatin from
D-cells, which inhibit both G-cells and enterochromaffin-like
cells. Hence, proton-pump inhibitors induce ­hypergastrinaemia.
It is usually recommended that inhibitors of acid secretion
should be stopped to measure and evaluate an elevated concen-
tration of gastrin in plasma. The utility of measuring intragastric
pH is often overlooked; hypergastrinaemia cannot be due to the
medication if gastric acid secretion is not suppressed.
The D-cell is also an intermediary in the enterogastrone mech-
anisms, and expression of the somatostatin gene appears to be
downregulated in Helicobacter pylori antritis.
Proton-pump inhibitors: given that only the proton pump is
­common to acid secretion, it is not surprising that its inhibi-
tors have transformed the management of acid-related disease.
Anticholinergics are readily bypassed and not of value clinically,
whereas H2-receptor antagonists and even vagotomy leave a sub-
stantial proportion of acid secretion intact. Gastrin receptor antag-
onists are in development. Acid is secreted with an osmotically
appropriate volume of water, and so proton-pump inhibitors also
reduce the volume of gastric juices, not just their acidity. This
contributes to their effectiveness in gastro-­oesophageal reflux
disease and also their adjunctive use in short bowel with gastric
hypersecretion.
Conceptual model: transepithelial signalling by EECs
Nutrients
Paracrine/endocrine
factors
EEC, enteroendocrine cell
Figure 1
SURGERY 27:6
Enterochromaffin-like cell hyperplasia
In addition to its role in secretion of gastric acid, gastrin is also
a direct growth factor for the enterochromaffin-like cell, which
explains the presence of enterochromaffin-like hyperplasia seen
in some chronically hypochlorhydric and consequently hyper-
gastrinaemic patients (e.g. in pernicious anaemia, in which there
is autoimmune destruction of parietal cells). This can progress
to small carcinoid nodules in a minority, and invasion and
metastasis can occur in a very small minority. This underlies the
rationale for antrectomy rather than total gastrectomy for corpus
carcinoids, removing the anatomical source of gastrin. The risk
of surgery appears higher than the risk of invasiveness and sur-
veillance is adequate initially. The risk of aggressive neoplasia
is higher in non-hypochlorhydric hypergastrinaemia (Zollinger–
Ellison syndrome and/or multiple endocrine neoplasia (MEN1).
Measuring intragastric pH is very helpful.
Biology of the intestinal epithelium
Gastrointestinal epithelial cells originate from a stem cell popula-
tion in the crypt zone. There are four cell types resulting from
differentiation pathways controlled by a complex array of tran-
scription and differentiation factors. The key lineage commitment
decision is whether to adopt the dominant pathway to an absorp-
tive phenotype (enterocyte/colonocyte) or a secretory phenotype.
This includes mucus-secreting goblet cells, hormone-secreting
enteroendocrine cells (EECs) (Figure 1), and defence peptide-
secreting ­Paneth cells. Progenitor cells originating from the stem
cell population differentiate along an absorptive (enterocyte) or
Lumen
Apical
Basolateral
Neurones
Epithelia
Muscle
Immune cells
226 © 2009 Published by Elsevier Ltd.
 Basic Science
secretory (EEC, Paneth cell, goblet cell) cell pathway under the
control of specific differentiation and transcription factors.
In the small intestine, the cell types, except Paneth cells,
ascend the crypt–villus axis, moving over a period of 3–5
days to be shed by apoptosis. Paneth cells move to the base
of the crypts, and appear to have a longer lifespan. Increas-
ing evidence implicates Paneth cells in the pathogenesis of
inflammatory bowel disease, given their key role in epithelial
recognition and defence against microorganisms. The Crohn’s
gene, CARD15, encodes a Paneth cell protein. Abnormal epi-
thelial structure in disease reflects changes in the regulation of
epithelial turnover. Some of this may be adaptive; for example,
increased turnover and goblet cell hyperplasia in response to
nematode infection may contribute to parasite expulsion (‘weep
and sweep’ hypothesis).
The epithelium as a barrier
The gut prevents the passage of bacteria and other undesirable
substances (dietary contaminants, bacterial products) from the
lumen into the organism. The colon contains tenfold more bac-
teria than cells in the host body. This is mainly achieved by tight
junctions between cells (Figure 2). These are complex structures
comprising multiple proteins that constitute a pore close to the
apical surface of the cells that filters molecules according to size.
Key members are ZO-1, occludin and the claudin family. This
constitutes the paracellular pathway, and is a minor route for the
absorption of some small ions (e.g. calcium). Water also passes
this way, with some movement occurring transcellularly. Increas-
ing interest has focused on the regulation of tight junctions, and
whether they contribute to the increase in intestinal permeability
seen in injury and inflammation in the gut. Current research aims
to identify factors that protect or restore the barrier, for example
antioxidants and nutrients (e.g. glutamine). The gut microflora
has an active symbiotic role in maintaining the barrier. Using pro-
biotic bacteria to alter bacterial flora and enhance the barrier has
Absorption of glucose, galactose, salt and water
Lumen
Glucose and galactose absorbed
from the lumen via SGLT-1
SGLT-1 H2O
Tight
junction
Na+
Glucose
Galactose
H2O
Na+
GLUT-2
Glucose and galactose exported K+
through the basolateral
membrane via GLUT-2
Basolateral membrane
SGLT-1, sodium-dependent glucose and galactose transporters; GLUT –2, glucose transporters; cAMP, cyclic AMP;
VIP, vasoactive intestinal peptide.
Figure 2
SURGERY 27:6
generated interest. Another approach is to give these with bacte-
rial nutrients (prebiotics); the combination is termed a ‘synbi-
otic’. A class of dietary fibre substances, fructo-oligosaccharides,
has also been shown to modulate permeability, an effect also
observed in germ-free (gnotobiotic) states. Changes in inflamma-
tory signalling by epithelial cells occur in response to probiotic
bacteria, suggesting an active intrinsic effect of fibre (previously
thought to be inert and solely the target of bacterial fermenta-
tion). There is also evidence that psychological stress increases
gut permeability via these or other structures. Increased perme-
ability leads to inappropriate fluxes of fluids and electrolytes, and
may underpin bacterial translocation, prequelling sepsis.
The mucosa is immunologically active. Defence against
injury is provided by secretory immunoglobin and various cell-
­mediated mechanisms, and sampling antigenic content via spe-
cialized dendritic cells scattered throughout the gut. These can
open tight junctions, passing processes between epithelial cells
to sample luminal contents.
Enteroendocrinology
The gut is the largest endocrine, with up to 20 types of EEC
scattered throughout the gastrointestinal epithelium. As noted
above, EECs are derived by selective terminal differentiation
from a common stem cell niche. EECs serve a variety of physi-
ological roles, but their key function is to operate as transepithe-
lial signal transduction conduits. The apical surface of most EECs
is ‘open’ to the lumen, projecting microvillus processes that are
believed to operate as chemosensors. Variables sensed intralumi-
nally include nutrients, pH and osmolarity. Each EEC produces
one or more regulatory peptide (or biogenic amines, principally
histamine and 5-hydroxtryptamine) which are secreted predomi-
nantly basolaterally by exocytosis. The released mediators were
thought to act as true hormones (via the circulation to act at
a distance) but many of their actions occur locally (paracrine
effects). The epithelium is a target, for example, in the regulation
Cholera toxin
cAMP
VIP
Cl- transported via a Cl- channel
activated by cAMP and VIP
Na+
2Cl–
K+
Sodium is required for the
Enterocyte transportation of
monosaccharides into both the
cytosol of the enterocyte and into
Na+
the basolateral space before
K+ entering the portal circulation
Na-K ATPase
227 © 2009 Published by Elsevier Ltd.
 Basic Science
of local secretomotor events, but afferent nerve fibre terminals,
particularly fibres of vagal origin, appear to be the major site of
action for many enteroendocrine factors.
The key difference between EECs and other endocrine organs
(e.g. pituitary gland, islets of Langerhans) is that the former exist
as individual cells scattered throughout the epithelium. This has
posed a major hurdle in studying these cells because there is no
method for isolating EECs and studying their function.
The key endocrine cells of the stomach have been discussed
in relation to acid secretion, but two other hormones from the
stomach have major roles. Leptin, the ‘fat controller’ originally
isolated from adipocytes, is also secreted by the pepsinogen-
secreting chief cells of the stomach (which were not previously
thought to have an endocrine nature), and to activate vagal
afferent nerves to contribute to satiety. Ghrelin is secreted by
a population of endocrine cells. This was originally identified
as a growth hormone-releasing (GH-Relin) factor, not an effect
believed to be mediated from the stomach. Ghrelin is unusual
in being the first gut hormone described that rises in the plasma
during fasting and falls upon feeding: it is unclear whether the
rising level pre-prandially is a signal to eat, or whether the falling
value after a meal constitutes a satiety signal. Ghrelin accelerates
gastric emptying, and is being studied in models of gastropare-
sis (e.g. diabetes). Reports that altered concentrations of ghrelin
after gastric bariatric and bypass surgery contribute to the value
of the procedure have been very inconsistent. Ghrelin and leptin
may also contribute to gastric mucosal protection.
The duodenum is a major enteroendocrine territory, with
immediate sampling of just-emptied gastric contents serving to
modulate the secretory and motility patterns controlling digestion
and absorption with maximal efficiency. Secretion of lipid-induced
CCK by the I-cell subtype of EECs triggers pancreatobiliary secre-
tions. CCK also delays the emptying of lipid-rich chyme from the
stomach, in addition to limiting further food intake by inducing
satiety (Figure 3). These effects of CCK are mediated largely by
vagal reflexes. The CCK-1 receptor is expressed by vagal afferent
neurones. The cell bodies lie in the nodose ganglion in the neck,
and the synthesized receptors are transported down the axono-
plasm to peripheral terminals where they are activated by CCK.
Recent work suggests that the vagal circuitry responds to sev-
eral factors inducing satiety (CCK, leptin, possibly cytokines) and
hunger (endocannabinoids, ghrelin), and integrates these positive
and negative signals in the short-term control of food intake. CCK
has also been implicated in the hypophagic state associated with
intestinal inflammation; CCK cell hyperplasia and hypersecretion
appear to contribute to the reduction in food intake observed.
Free fatty acids rather than intact triglyceride induce secretion
of CCK (hence lipase inhibitors such as orlistat may blunt the
satiating effects of meals). Secretion of CCK is also impaired in
pancreatic insufficiency. The molecular basis of fatty acid sensing
by EECs is unclear, but the recent identification of four fatty acid
receptors (G protein-coupled receptor (GPR) 40, 41, 43 and 120)
has yielded candidate mechanisms and potential pharmacologi-
cal targets. The best characterized is GPR40, responsible for fatty
acid-induced secretion of insulin by pancreatic β-cells.
Secretin cells respond to acidic pH and fatty acids to induce
pancreatic alkaline secretions. Another key cell type, the L- cell,
secretes glucagon-like peptides-1 and -2 and peptide YY. ­Glucagon-
like peptide-1 also mediates delayed gastric and ­intestinal transit,
SURGERY 27:6
Response to a fatty meal
Fatty acids
Mucosal
EEC
Basolateral
CCK
Pancretic GI motility Gallbladder Satiety
exocrine emptying
secretion
In response to a fatty meal, cholecystokinin (CCK) release coordinates
responses including regulation of pancreatic exocrine secretion and control
of gastrointestinal (GI) motility, in particular gallbladder emptying and gastric
emptying; it has now been recognized as an important satiety factor.
EEC, enteroendocrine cell
Figure 3
whereas glucagon-like peptide-2 is implicated in epithelial tro-
phism and repair (this underpins its evaluation in the therapy of
intestinal failure and short bowel). Glucagon-like peptide also has
an ‘incretin’ effect, signalling to the pancreas to induce insulin
secretion in the absence (but anticipation) of a rise in blood glu-
cose. Peptide YY responds to nutrients, particularly fat, arriving
in the terminal ileum; this heralds imminent malabsorption and
hence nutrient wastage, and triggers the ‘ileal brake’ mechanism,
further delaying gastrointestinal transit.
The other key endocrine cell of the gut is the ­enterochromaffin
cell, whose major product is the amine 5-hydroxytryptamine.
About 97% of the 5-hydroxtryptamine in the body is in the
gut, and its release regulates motility and secretion throughout
the intestine. Increased numbers of enterochromaffin cells and
secretion of 5-hydroxtryptamine have been reported in gut infec-
tion, but this appears to persist after resolution of infection, and
may be a component of the functional gut symptoms frequently
observed following enteritic episodes. Increased numbers of
enterochromaffin cells have been reported in post-infectious irri-
table bowel syndrome. There is little other evidence of disorders
of the enterochromaffin system, other than rare tumours.
Gastrointestinal motility
‘Motility’ is the term used to describe the orderly processes that
move the luminal contents from the mouth to the anus. The
dominant process in the oesophagus and small bowel is peri-
stalsis, in which a bolus is propagated by a wave of contrac-
tion. Peristalsis is an intrinsic property controlled by the neural
plexus, and persists in extrinsically denervated gut (Figure 4).
228 © 2009 Published by Elsevier Ltd.
 Basic Science
Peristalsis in the small intestine
The muscles behind the bolus of food
contract, while the ones in front relax,
which moves the bolus along in the
direction of the arrow. Peristalsis is
controlled by the intrinsic neural plexus
network. Excitatory motor fibres
releasing ACh and substance P cause
contractions, while inhibitory motor
fibres release VIP and NO. Mucosal wall
receptors detect the food bolus and
interact with the excitatory and
inhibitory fibres to either increase or
decrease contraction
ACh, acetylcholine;
VIP, vasoactive intestinal peptide;
NO, nitric oxide.
Figure 4
The intrinsic rhythm appears to be generated by specialized
neurones called the interstitial cells of Cajal, which govern
the activity of local smooth muscle. These neurones express
the protein c-kit, and are therefore the likely cell of origin of
gastrointestinal stromal tumours which are ­ characterized
­immunohistochemically by c-kit positivity. Recent support-
ing data have suggested that gastrointestinal stromal tumour
cells retain some of the electrophysiological properties and ion
channels typical of the interstitial cells of Cajal. Data also are
accumulating for loss of interstitial cells of Cajal in disorders of
gastrointestinal motility, particularly slow transit constipation
with acquired megacolon, but also in acute obstruction, Chagasic
megacolon and diabetic gastroenteropathy. It is however possible
that interstitial cells of Cajal are lost as a secondary consequence
of the motility disorder.
Gastrointestinal motility is largely an intrinsic property of the
gut, but is subject to external influences. In general, the parasym-
pathetic (vagal and sacral) pathways increase motility via post-
ganglionic fibres utilizing acetylcholine, substance P and ATP.
Sympathetic noradrenergic spinal fibres tend to inhibit motility;
inhibitory α2-receptors are expressed on post-ganglionic vagal
fibres and reduce cholinergic transmission. Hormones also affect
motility. CCK inhibits gastric and small bowel motility, but stim-
ulates the colon, and may be responsible for the gastrocolic reflex
(in which eating can trigger an urge to defaecate). Thyroid hor-
mones are stimulatory. Glucagon and opioids have strong anti-
motility effects in the gut. Electrolyte disturbances (particularly
K+ and Ca2+) can also have profound effects on neuromuscular
function. Congenital or acquired abnormalities of visceral muscle
or the enteric nervous system are likely to underlie the pseudo-
obstructive syndromes. A wide range of common drugs is also
able to influence motility.
Gastric motility: the pattern of motility is quiescent initially
(phase I) in the fasting state. After about 40 minutes, activity
restarts (phase II), with a gradual increase in contractions that
SURGERY 27:6
Contraction Relaxation
ACh VIP
Substance P Mucosal NO
and wall
receptors
+ +
Myenteric
plexus
Excitatory motor fibre Inhibitory motor fibre
begin in the stomach and reach a peak of intensity (phase III)
lasting about 10 minutes, before returning to phase I quiescence.
This phase III pattern starts in the stomach (‘hunger contrac-
tions’) and travels along the small bowel over about 90 minutes; it
is termed the migrating motor complex. This acts as an ‘intestinal
housekeeper’, sweeping out the small bowel to prevent stagna-
tion and bacterial contamination. Gastric, biliary and pancreatic
secretions are also triggered by the migrating motor complex,
which is coincident with a peak in circulating motilin. This hor-
mone is mimicked by erythromycin, a prokinetic antibiotic.
Feeding interrupts this pattern. The proximal stomach under-
goes tonic relaxation via a vagal reflex, with further phasic
relaxations. This allows the intragastric volume to rise without
a commensurate increase in pressure. The loss of such ‘adaptive
relaxation’ may partly contribute to the early fullness and rapid
gastric emptying seen after vagotomy. In the fed state, rhythmical
contraction of the antrum at a rate of 3 contractions per minute
acts as a mechanical pump to emulsify food and, in coordination
with the pylorus, propel food into the duodenum. The pylorus
also acts as a sieve and relatively little food of greater than 3 mm
in diameter passes through.
Foods rich in lipids markedly slow gastric emptying. They
exert an inhibitory effect on the antral pump, stimulate pyloric
contractions and maximally relax the proximal stomach. These
effects are mainly mediated by CCK acting on CCK-1 receptors on
vagal afferent fibres.
The time taken for gastric emptying is highly variable and can
be up to 5 hours, depending on the type of nutrient, osmolal-
ity and temperature. Meals light in nutrients, and liquids, can
be emptied within 1 hour. Attempts to define normality must
be interpreted cautiously, but many patients with functional
­dyspepsia and early satiety lie outside the apparent norms.
Intestinal motility: the small intestine propagates waves at a
higher frequency than the antrum (about 12 contractions/­
minute) although peristalsis is also regulated by intrinsic reflexes
229 © 2009 Published by Elsevier Ltd.
 Basic Science
to distension (Figure 4). Small intestinal transit to the caecum
takes about 90 minutes.
The main function of the colon is water absorption, and
movement of the contents slows down. Bacteria are present and
the migrating motor complex dissipates at the ileocaecal valve.
Reflux of colonic contents into the terminal ileum triggers expul-
sive contractions to maintain relative sterility. Colonic transit may
take 24–48 hours, and occurs by haustration and mass move-
ment. Haustration comprises slow, segmental contractions over
several centimetres, and is responsible for the gross appearance
of the colon. Haustration mixes the colonic contents to facilitate
water absorption.
Mass movement involves episodic muscle contractions over
a longer segment of colon and occurs only a few times daily. It
resembles peristalsis in that the distal segment of colon relaxes
in anticipation, producing a wave that propagates at a rate of
SURGERY 27:6
about 1 cm/second to move the colonic contents distally. Their
arrival in the sigmoid colon leads to an urge to defaecate, and
an increase in amplitude has been noted in some patients with
irritable bowel syndrome. ◆
Further reading
Aziz Q, Thompson DG. Brain-gut axis in health and disease.
Gastroenterology 1998; 114: 559–78.
Champion MC, Orr WC, eds. Evolving concepts in gastrointestinal
motility. Oxford: Blackwell Science, 1996.
Dockray GJ. Gastrin and gastric epithelial physiology. J Physiol 1999;
15: 315–24.
Smout AJMP, Akkermans LMA. Normal and disturbed motility of the GI
tract. Stroud: Wrightson Biomedical, 1992.
230 © 2009 Published by Elsevier Ltd.