Re: Re: to - re: trypsinogen and amalyse tests..

December 21, 2001

Serum Complex of Tryspin

Serum Complex of Trypsin-2 and a 1-antitrypsin: A New Sensitive Marker of

Acute Pancreatitis

Johan Hedstr?m, M.D*., Jari Leinonen, M.Sc., and Ulf-H?kan Stenman, M.D.

Departmetnt of Clinical Chemistry, Helsinki University Central Hospital,

Helsinki, Finland

Introduction: Pathological intrapancreatic activation of trypsinogen to

trypsin occurs in acute pancreatitis (AP). When reaching blood, trypsin-2

forms a complex with a1-antitrypsin (AAT). The trypsin-2-AAT complex can be

specifically measured by a recently developed double antibody sandwich

assay.

Purpose: To estimate the diagnostic and prognostic accuracy of serum

determinations of trypsin-2-AAT in AP. Serum CRP, amylase and trypsinogen-2

were used as reference methods.

Design: A retrospective study on consecutive patients during March 1992 to

November 1993.

Setting: Patients treated for AP and other acute abdominal disorders at the

Second Department of Surgery at Helsinki University Central Hospital.

Methods: 110 patients with AP and 66 patients with acute abdominal diseases

of extrapancreatic origin were studied. The final diagnosis of AP was based

in findings of upper abdominal pain accompanied by the typical appearance of

AP in ultrasonography or computed tomography (CT). Based on the clinical

course, AP was classified as mild (n=82) or severe (n=28). Trypinogen-2 and

trypsin-2-AAT were determined by time- resolved immunofluorometric assays

(IFMA). The upper reference limit was 12 ?g/L. The ability of various tests

to differentiate between mild and severe AP and nonpnacreatic disease was

estimated on the basis of sensitivity and specificity at clinically relevant

cut-off levels and the validity of the test was further evaluated by

receiver-operating characteristic (ROC) curve analysis.

Results: At admission, all patients with AP had clearly elevated values of

trypsin-2-AAT (= 32 ? g/L), whereas only 5% of the controls had such values.

In AP, trypsinogen-2 and trypsin-2-AAT increased earlier than CRP and

remained elevated longer than amylase. There was also less overlapping

between patients with AP and controls for trypsin-2-AAT than for the other

markers. Time course profiles of trypsin-2-AAT showed that in severe cases

it mostly peaked in the initial sample and slowly decreased during the next

days. In patients with mild AP the peak was mostly observed in the second

day. Of the markers studied, trypsin-2-AAT showed the best accuracy (largest

area under the ROC curve) both in differentiating AP from controls and mild

from severe disease. At presentation, trypsin-2-AAT differentiated between

mild and severe AP much more accurately than CRP, AUC being 0.82 and 0.73,

respectively.

Conclusion: Of the markers studied, trypsin-2-AAT displayed the best

accuracy for differentiating between AP and extrapancreatic disease as well

as for predicting a severe course of the disease at presentation. If

available on automated instrumentation and on emergency basis, the assay

could markedly improve the diagnosis of this common and potentially lethal

disease.

Mark E. Armstrong

www.top5plus5.com

Oregon State Chapter Rep

Pancreatitis Association, International

Re: Re: to - re: trypsinogen and amalyse

tests..

> In 1996, I was seen at The Cleveland Clinic where I learned about blood

> trypsinogen. You see, amylase and lipase levels are not the only

indications

> of a sick pancreas.

>

> The blood sample was drawn in Ohio, but sent to a lab in Utah:

>

> Associated Regional and University Pathologists, Inc. or ARUP

> 500 Chipeta Way

> Salt Lake City, UT 84108

>

> They have a toll-free number - 800 242-2787.

>

> At that date, the accepted normal trypsinogen range was

> 13.0-36.0 ng/mL

>

> The expected values for:

>

> Chronic Pancreatitis...less than 13.0 ng/mL

> Acute Pancreatitis... greater than 45.00 ng/mL

>

> My result was 10.2.

> And I finally got my diagnosis.

>

> There are probably other labs around the country who perform the test. If

> your doctors don't know about them, then you folks must find them. And

the

> Internet is where to look.

>

> -E

>

> PANCREATITIS Association, Intl.

> Online e-mail group

>

> To reply to this message hit " reply " or send an e-mail to:

PancreatitisYahoogroups

>

> To subscribe to this e-mail group, simply send an e-mail to:

Pancreatitis-subscribeYahoogroups

>

December 21, 2001

SECRETION OF THE PANCREAS, GALLBLADDER AND LIVER

Secretion

The Pancreas weighs 100g, but it secretes over 10 times its weight in one

day. It has both an endocrine and exocrine function. The endocrine function

is primarily in the islets of Langerhans which have four types of cells:

insulin secreting (60-80% of total), glucagon secreting (15-20%),

somatostatin and pancreatic polypeptides. The exocrine pancreas structure

has acinar and ductal cells. The acinar cells make the digestive enzymes and

the duct cells make the aqueous component. The acini are organized around

the terminal end of a duct with zymogen granules facing the lumen. The

insular-acinar axis is the connection between the exocrine and endocrine

pancreas. The islet cells have arterioles but few venules. Almost all of the

blood that comes into the islets goes on to the acinar tissue. These acinar

cells are dependent on insulin for growth. Insulin also promotes secretion

from acinar cells when the acinar cells are stimulated by other hormones.

The ductal cells are responsible for the large volume of bicarbonate rich

fluid which is in pancreatic juice. This fluid is added to the enzymatic

fluid that was secreted from the acinus as it flows down the intralobular

duct. At both a slow and fast flow rate of pancreatic juice the

concentration of both sodium and potassium is equal to that of plasma.

However bicarbonate concentrations increase with increasing flow rate and

chloride concentrations reciprocally decrease at higher flow rates. This

ensures that at all flow rates the final tonicity of pancreatic juice will

always be equal to that of plasma. The bicarbonate secreted by ductal cells

is predominantly derived from plasma (93%). The process involves an Na/K

ATPase, a Cl/HCO3 antiporter, carbonic anhydrase and depends on the

intracellular chloride concentration. This is kept low by a cAMP controlled

chloride channel at the apical border of both ductal and acinar cells. In

cystic fibrosis this Cl channel (called CFTR) is defective and doesn't

respond to cAMP which causes mucus plugging of the pancreatic duct. Ductal

cells are very sensitive to secretin and VIP hormones, both of which

increase cAMP levels. CCK has little effect on bicarbonate secretion from

ductal cells when acting alone but it can potentiate the effect of secretin.

The digestive enzymes of pancreas are involved in digestion of large complex

molecules. They include the proteinases: trypsinogen, chymotrypsinogen,

procarboxypeptidase and proelastase. It also produces pancreatic amylase

(for carbohydrate breakdown), lipase (lipid breakdown), procholipase I and

II and prophospholipase A2. Trypsinogen is cleaved to form trypsin at a

lysine AA. This process is initiated in the intestine by enterokinase which

activates trypsin to cleave the other trypsinogens. Trypsin also can

activate all the other pancreatic proenzymes. The enzymes are all secreted

in a nonactive form to prevent autodigestion of the secreting cells. The

body has numerous mechanisms to prevent autodigestion:

All digestive enzymes except lipase and amylase are synthesized in an

inactive form Enterokinase, the activating enzyme of the trypsin cascade, is

physically separated from trypsin since it is found in the small intestine.

The digestive enzymes within the acinar cells are packaged into zymogen

granules. The intracellular Ca concentration is kept low which prevents

trypsinogen activation. Acinar cells synthesize pancreatic secretory trypsin

inhibitor (PSTI) which inactivates trace levels of trypsin. If excessive

levels of trypsin build up in the cell it will autodigest itself, thereby

inactivating itself. The liver produces two inhibitors which are found in

the blood, alpha1 antitrypsin and beta2 macroglobulin which inhibit any

trypsin found in bloodstream.In one form of hereditary pancreatitis, there

is a genetic defect in one of the trypsinogen genes which loses the ability

of trypsin to inactivate itself at very high levels. Intracellular calcium

is the most important determinant of pancreatic secretion. It is also

stimulated by elevated cAMP. The nervous system stimulates secretion via

parasympathetics from the vagus which have cholinergic receptors on the

cells. The sympathetic nervous system inhibits pancreatic secretion.

Acetylcholine and gastrin releasing peptide are the neurotransmitters which

the vagus uses to stimulate secretion. They activate Ca dependent secondary

messengers (PLC, IP3, DAG) which results in the release of zymogen granules.

VIP is a neuropeptide used by intrapancreatic nerves which increases the

levels of cAMP and accentuates the effects of ACh. CCK and secretin are two

hormones released from the duodenum which stimulate pancreatic secretion

after ingestion of a meal via cAMP. CCK probably modulates its effect via

stimulation of vagal nerves. There are 3 phases of pancreatic stimulation:

cephalic- initiated by sight and smell of food and mediated by vagus.

Accounts for 25% of pancreatic secretion during a meal. Gastric- caused by

gastric distention via vagal-vagal reflex. 10% of pancreatic secretion.

Intestinal- caused by food in the intestine eliciting the secretion of CCK

and secretin into duodenum and by stimulation of vagal fibers.Duodenal

acidification causes release of secretin and stimulates the vagus to

activate ductal cells to release a bicarbonate rich fluid. Proteins

stimulate the secretion of CCK into blood which causes the acinar cells to

secrete proteinases. Inhibitory hormones include somatostatin, pancreatic

polypeptide, and peptide YY. Mostly these inhibit cholinergic nerves.

Somatostatin is the most potent.

LIVER AND GALL BLADDER Each lobule of the liver is organized around a

central vein and blood flows from the periphery, from the hepatic artery and

the sinusoids. Because of fenestrations in the endothelial cells which line

the sinusoid, each hepatocyte is in direct contact sinusoidal blood. Biliary

canaliculi lie between adjacent hepatocytes and bile flows in the reverse

direction of the blood. The canaliculi drain into bile ducts at the

periphery of the lobules. The liver performs multiple functions: regulates

metabolism of carbohydrates and lipids, synthesizes proteins and other

molecules, stores vitamins and iron, degrades hormones and inactivates and

excretes drugs and toxins.

Carbohydrates: when the level of glucose in the blood is high, some of the

glucose is converted into glycogen, which is then deposited into the liver.

When the blood glucose level is low, glycogen in the liver is broken down to

glucose (glycogenolysis) which then gets released into the blood stream. The

liver also has the ability to perform gluconeogenisis, the conversion of

amino acids, lipids, or simple carbohydrates into glucose.

Lipids: Absorbed lipids leave the intestines in chylomicrons in the lymph.

Along the way it is converted and changed into chylomicron remnants which

are rich in cholesterol. These remnants are taken up by the hepatocytes and

degraded. The hepatocytes also synthesize and secrete VLDL. VLDLs are then

converted into other types of serum lipoproteins, HDL or LDL. These

lipoproteins are the major source of cholesterol and triglycerides that

supply most other tissues of the body. The liver is also involved in bile

synthesis and bile is the only route of excretion of cholesterol from the

body. So the hepatocyte is the principal source of cholesterol for the body

while also the major site of excretion of cholesterol.

Proteins: When proteins break down by catabolism, some of the amino acids

deaminate and form ammonia. The ammonia can not be further broken down by

most tissues and toxic levels of ammonia can accumulate. The ammonia can be

dissipated by conversion to urea which takes place mainly in the liver. The

liver also synthesizes all the non-essential amino acids. It also

synthesizes all the major plasma proteins, which includes the lipoproteins,

albumins, globulins, fibrinogens, and other proteins involved in blood

clotting.

Storing Vitamins: These vitamins include A, D, and B12 so that during

transient deficiencies it can protect the body. Next to hemoglobin in the

RBC's, the liver is the most important storage site of iron. The liver

transforms, breaks down and excretes many hormones, drugs and toxins.

The liver's role in digestion is with the secretion of bile. Bile, produced

by hepatocytes, contains bile acids, cholesterol, phospholipids, and bile

pigments. All of these constituents are secreted by hepatocytes into the

bile canaliculi, along with an isotonic fluid that resembles plasma in its

electrolyte concentrations.

The secretory function of the liver resembles that of the exocrine pancreas.

In both the primary secretion is isotonic to plasma and the levels of Na, K,

and Cl are close to plasma levels. When stimulated by secretin, the ductular

epithelial cells contribute an aqueous secretion with a high bicarbonate

concentration. So, similar to pancreas, in both the ducts change the

isotonic to plasma solution and add bicarbonate.

Between meals, bile is diverted into the gallbladder. The gallbladder

epithelium extracts salts and water from the stored bile and concentrates

the bile acids fivefold to twentyfold. Cholocystokinin (CCK) is the most

potent stimulus for emptying of the gallbladder. Bile acids emulsify lipids

and thereby increase the surface area available to lipolytic enzymes. Bile

acids then form mixed micelles. Micelles increase the transport of the

products of lipid digestion to the brush border surface. The epithelial

cells (intestines) actively absorb bile acids, mainly in the terminal ileum.

Only about 10-20 percent of bile acids escape absorption and are excreted.

What happens to the rest, is that bile acids return to the liver and are

taken up again by the hepatocytes. This concept is known as enterohepatic

circulation- that the entire bile acid pool is recirculated two or more

times in response to a typical meal.

About 60% of bile is bile acids (Cholate, deoxycholate, chenodeoxycholate,

lithocholate), 22% phospholipids, while smaller composition including

cholesterol and bile pigments. Bile acids make up 65% of dry weight of bile.

Primary bile acids are the major bile acids synthesized by the liver (cholic

acid and chenodeoxycholic acid). The presence of the carboxyl or hydroxyl

groups added (after it started as cholesterol) makes it more of a polar

compound. Secondary bile acids are deoxycholic acid and lithocholic acid.

When bile acids are released they are amphipathic in that there is a

hydrophobic and hydrophilic side. When they create micelles the hydrophilic

side faces outward and the hydrophobic side faces inward, allowing the

lipids to accumulate inside. Bile acid micelles form when the concentration

of bile acids exceeds a certain limit, the critical micelle concentration.

Hepatocytes also secrete phospholipids and cholesterol in bile. The

predominant phospholipid is lecithin. One of the major routes for

cholesterol to excreted is into the bile. The secretory mechanism into the

bile canaliculi of the phospholipids and cholesterol involves exocytosis.

When aging RBC's are degraded in the reticuloendothelial system, the

porphyrin moiety of hemoglobin is converted into bilirubin. Bilirubin is

released into plasma where it is bound by albumin. The bilurubin is

glucoronidated in the liver into bilirubin glucoronide and that is secreted

into the bile.

Secretion of bile duct epithelium: The epithelial cells that line the bile

ducts secrete an aqueous secretion that accounts for 50% of total volume of

bile. This secretion is isotonic and contains Na and K concentrations

similar to plasma. However, the concentration of bicarbonate is greater and

the concentration of chloride is less then that in the plasma.

Gallbladder and bile concentration: Between meals, the tone of the sphincter

of Oddi, which guards entrance of the common bile duct into the duodenum, is

high and diverts the bile flow into the gallbladder. The gallbladder has

capacity to hold 15-60 cc of fluid, in humans. The gallbladder concentrates

the bile acids as it absorbs the sodium and releases it out the lateral side

while chloride and bicarbonate gets driven with it. When that happens the

water follows it out as well. This allows the gallbladder to concentrate its

contents from 5 to 20 fold. (The " standing osmotic gradient mechanism for

fluid absorption was first proposed for the gallbladder).

Emptying of gallbladder begins several minutes after the start of a meal.

Intermittent contractions of the gallbladder force bile through the

partially relaxed sphincter of Oddi. During the cephalic and gastric phases

of digestion, gallbladder contraction and relaxation of the shincter are

mediated by cholinergic fibers in the vagus nerve and by gastrin released

from the stomach. The highest rate of gallbladder emptying occurs during the

intestinal phase of digestion, with the strongest stimulus for this emptying

being CCK.