Tag Archives: ultrasound

neutropenia and garlic

All cancer chemotherapy is unpleasant, but the chemo we use for breast cancer is particularly nasty. Megan is 48 and was admitted this week with a fever and diarrhoea, a week after her third course of CEF – cyclophosphamide, epirubicin and 5-flurouracil.  Her breast cancer was diagnosed a few months ago – she noticed a lump in her left breast when washing herself in the bath. Megan could tell from the face of her GP when she was examined that it was going to be bad.

Then, shortly after, there was the ultrasound, mammogram and trucut needle biopsy at the breast clinic which hurt more than she was expecting – Megan was really scared by this time.

mammogram of breast showing a cancer bottom right
mammogram of breast showing a cancer bottom right

She remembers vividly the meeting with the breast surgeon a week later, but only as a jumble of “surgery”, “partial mastectomy”, “total mastectomy”, “node clearance”, “chemotherapy”, “radiotherapy”. Her husband Jack was with her, but it didn’t make much sense to him either. They went home with a pile of leaflets and phone numbers.

The surgery went fine, everyone was really nice and caring. Then a meeting with the oncologist. She advised chemotherapy then radiotherapy. That seemed much more simple. But chemotherapy was awful. Megan felt sick and hopeless. All she could do was sit in bed and do crosswords. Just as she was starting to feel better the next round of chemo was due.

epirubicin is an anthracycline antibiotic chemotherapy agent which works by damaging DNA
epirubicin is an anthracycline antibiotic chemotherapy agent which works by damaging DNA

When I met her she had the look of a usually bright and cheerful person who was wondering if she could take any more. I told her that her white cell count was low, and we needed to give her intravenous antibiotics until her fever resolved and white cell count recovered.

How does cancer chemotherapy work? Why does it kill cancer cells in preference to normal cells in our body?

The normal answer I get from the young doctors and students is that chemotherapy targets rapidly-dividing cells. It really is not quite as simple as that. Breast cancer cells divide much less rapidly than bone marrow or gut epithelial cells, but chemotherapy is designed to kill all the cancer cells and leave our bone marrow and intestinal lining intact. To answer the question properly, we need to understand what makes cancer cells malignant.

We have a much better understanding of the molecular basis of cancer than we did thirty years ago. Cancer cells usually have a number of genetic abnormalities which result in the cells no longer behaving in a useful and regulated way. Cells, whether they are from breast, colon, lung or any other tissue need to behave in a regulated and responsible way. They need to co-operate with the cells around them, working together to make sure the organ works properly. Like in normal society, where people have to abide by rules which make sure everything works properly. If someone misbehaves badly, the police force and courts act to stop them doing the bad thing. Similarly, if a cell is damaged and behaves abnormally, the body has ways of making sure that is either removed or repaired. Our immune system is constantly removing abnormal cells, and all cells have a mechanism to detect and repair DNA damage. One of the main mechanisms to identify DNA damage is the p53 system. The p53 gene is turned on when DNA damage is detected. This causes p53 protein to be made and cause the cell to stop dividing until the DNA damage is repaired. If the DNA cannot be repaired the cell undergoes programmed cell death. Harsh, you may think, but necessary to prevent the cell dividing and making more abnormal cells – “here is a pistol – I think you need to go to the library and consider your options”.

When cells have damaged DNA which cannot be repaired they do the decent thing
When normal (non-malignant) cells have damaged DNA which cannot be repaired they do the decent thing

The problem comes when the law enforcers are compromised. Corruption in the police force or judges is disastrous for society and for our bodies. Most common cancers have damage to the p53 gene, meaning that cells will divide even when DNA is damaged. Understanding this helps us understand why traditional chemotherapy targets cancer cells.

Cyclophosphamide is a nitrogen mustard alkylating agent which damages DNA by permanently crosslinking the two DNA strands. Nitrogen mustard has been molecule of the month: find out more about its chemisty here:

http://www.bris.ac.uk/Depts/Chemistry/MOTM/mustard/mustard.htm

In normal cells this damage is detected and prompts an activation of the p53 gene causing production of p53 protein –  with consequent shutdown of cell division until the damage is repaired (or, if the damage cannot be repaired the cell commits sucicide). In cancer cells which cannot make p53, the cells will continue to divide and render them more susceptible to further DNA damage. This is because when cells are dividing the DNA is unravelled and more exposed to the damaging chemotherapy agent.

Continued division of cancer cells will also make them more susceptible to 5-fluorouracil. This drug inhibits the enzyme thymidylate synthase, preventing the production of normal thymine, one of the DNA bases. The cells are trying to divide but cannot make thymine and suffer a fate known as “thymineless death”.  Cells which have normal p53 will have shut down and stopped dividing, and will not suffer in this way.

Epirubicin is a member of a group of drugs called anthracycline antibiotics, derived from steptomyces bacteria. These substances also work by binding to DNA and interfering with cell division, again targeting the breast cancer cells which are still dividing, despite having their DNA damaged, because of defective p53.

This combination of drugs work well to kill the breast cancer cells, but the shutdown of normal bone marrow production and intestinal epithelial cell division still causes a major problem, as evidenced by Megan and her neutropenic sepsis and diarrhoea. Without white cells and gut epithelial cell production she cannot not defend herself against normal germs.

Patients undergoing chemotherapy are given a whole pile of leaflets including advice about neutropenic sepsis
Patients undergoing chemotherapy are given a whole pile of leaflets including advice about neutropenic sepsis

Damage to the p53 gene is not the only abnormality that causes breast and other cancers. Most breast cancer cells have a large number of genetic abnormalities, many of which we know are important in making the cells cancerous, but we do not know how. There is a really good video which explains what we know about the genetic abnormalities in breast cancer and other cancers by Prof Sir Mike Stratton FRS –

Also, it is being recognised that epigenetic abnormalities are common in cancer cells, with differences in DNA methylation between normal and cancer cells. As I mentioned in a previous post, epigenetic changes are likely to be important in ageing, as well as cancer. I think there maybe an important link between these.

What causes the DNA damage in breast cells?  That is a big problem to understand. It is easy to imagine how lung epithelial cells are exposed to cigarette smoke which contains carcinogens, and how colon epithelial cells may be damaged by nasty substances in our diet. But breast cells – why do they get damaged? The main risk factors for breast cancer are:

1 early menarche (onset of menstrual periods at an early age

2 late first pregnancy

3 fewer pregnancies

4 obesity

5 alcohol

How do these factors damage DNA and cause cancer? I do not know. Answers please.

Now on to the food link – garlic.

garlic has its characteristic odour because it contains the molecule allicin, which helps protect it from bacteria and insects which want to eat it
garlic has its characteristic odour because it contains the molecule allicin, which helps protect it from bacteria and insects which want to eat it

Cyclophosphamide is a nitrogen mustard. It is called that because mustard gas was thought to smell like mustard. In fact it smells more like garlic.

poster from first world war
poster from first world war – wikimedia commons

Garlic contains a substance known as allicin, which has a similar molecular shape to nitrogen mustard.

allicin and nitrogen mustard have a similar molecular shape - which is maybe why they have a similar smell
allicin and nitrogen mustard have a similar molecular shape – which is maybe why they have a similar smell

It has been suggested that garlic has all manner of wonderful properties to keep us healthy, including lowering cholesterol, preventing heart attacks and protecting us against infection (and vampires). The hard evidence is not there. But as Neils Bohr, the eminent physicist, said when a visiting journalist remarked “ why Professor Bohr, do you have a horseshoe above your front door – surely you cannot believe that it will bring you good luck?” replied:

“Of course I don’t believe in it, but I understand it brings you luck, whether you believe in it or not.”

That’s more or less what I think about garlic.

gallstones and mayonnaise

A few weeks ago we admitted Sylvia. She had been feeling unwell for a some days, with fever and episodes of uncontrollable shaking. What really alarmed her was when she turned yellow, and then developed abdominal pain and vomited a couple of times. Sylvia is forty eight, and had always been healthy. She rowed with the local club twice a week and kept her weight under control. For the past year or so she noticed that she got pains in her abdomen after eating fatty foods such as fish and chips, and now avoided foods like that. It was her son who noticed she was yellow – he then phoned Sylvia’s GP. The duty doctor came and confirmed that she was indeed jaundiced, feverish and unwell, and persuaded her that she needed to go to hospital.

these people look jaundiced - in fact they are not - the whites of their eyes are white - this image is copyrighted but Wiki thinks its ok to use it so I'm hoping its ok for me to do so
these people look jaundiced – in fact they are not – the whites of their eyes are white – this image is copyrighted but Wiki thinks its ok to use it so I’m hoping its ok for me to do so

We did all the usual things for someone with suspected biliary sepsis. Took blood cultures and routine blood tests. These tests confirmed she had elevated bilirubin and transaminases, increased alkaline phosphatase, as well as a high neutrophil count and CRP.  We gave her intravenous fluids, anti-emetics and antibiotics. We were able to get an ultrasound within a couple of hours. Ultrasound is really good at looking at the liver and biliary system in jaundiced patients – better than CT.

The ultrasound showed that Sylvia had a gallbladder full of gallstones and her common bile duct was dilated. It seemed likely that she had a stone blocking the flow of bile from the liver to the duodenum, and that the stagnant bile had become infected with bacteria causing ascending cholangitis and liver inflammation.

bile is made in the liver - it is stored in the gallbladder and squirted into the small intestine after a fatty meal, along with pancreatic secretions - Sylvia's gallstone was impacted in the sphincter of Oddi
bile is made in the liver – it is stored in the gallbladder and squirted into the small intestine after a fatty meal, along with pancreatic secretions – Sylvia’s gallstone was impacted in the sphincter of Oddi

The questions I want to answer this week are:

Why did Sylvia turn yellow?

What are gallstones made of and why do they form?

Why is there an increase in alkaline phosphatase in the bloodstream when the biliary system is obstructed?

The last of those questions is the hardest, but I will make some conjectures, and  hope that others who know more about it will correct me.

Jaundice is fairly straightforward. It is caused by the accumulation of bilirubin, which is a greenish-yellow colour. Bilirubin is made from haem, the central working part of myoglobin, but most bilirubin comes from the breakdown of haem in the haemoglobin from red blood cells.

So why is haemoglobin red? It is not only – or even mainly – the iron that makes haem red, but the porphorin ring that surrounds the iron. This large ring is made of four smaller pyrrole rings.

Pyrroles are often coloured – the dye indigo has two pyrrole rings and red acrylic paint often contains the dye pyrrole-red. Melanin, the pigment that gives skin and hair its colour is brown or black or red because it has a related structure – indole – a pyrrole stuck to a benzene ring. Without this pyrrole ring we would be like albinos, but without pink skin and eyes.  As we shall see, we would also produce beige poo and colourless urine.

this is a pyrrole - all pyrroles are highly coloured, and are in most of the things which make people the colour they are
this is a pyrrole – all pyrroles are highly coloured, and are in most of the things which make people the colour they are

Red blood cells last on average 120 days before they wear out and have to be recycled.  As we have five litres of blood and in each litre there is at least 120 grammes of haemoglobin, it means we have to make, and break down, five grammes a day.  About a teaspoonful.  This all happens inside macrophages in the spleen and liver, as they try to salvage useful parts of the red cells to make new things.

Macrophages are similar to neutrophils but slightly smarter.  Some are good at host defence: they come to the scene after the neutrophils have done their job, do the forensics, and find out what the germs are made of. They then tell the lymphocytes how to make antibodies to protect us in the future.

Other macrophages are good at clearing up the mess, breaking down dead tissue and making things neat and clean.  The spleen and liver macrophages that deal with old red cells are the latter kind. When they take red cells apart, they separate haem from globin, and then take the iron out of the haem.  Next, the porphorin ring is broken to form bilirubin – its molecule of linked pyrrole rings looks like four beach huts in a row.

haem is broken down by the enzyme haem oxygenase to form bilirubin
haem is broken down by the enzyme haem oxygenase to form bilirubin

The bilirubin is then thrown away (I don’t know why it can’t be re-used).  It attaches to albumin and becomes water soluble after conjugating in the liver with a sugary molecule called glucuronate.  The conjugated bilirubin is then transported into the biliary system, stored in the gallbladder, and squirted into the duodenum when we eat fatty food.  In the bowel, it is converted to urobilinogen, which makes poo brown.  Some is reabsorbed and excreted in urine, which makes urine yellow.Slide2

Sylvia had noticed that her poo had changed colour – it had become putty-coloured – and her urine had become much darker. That is because conjugated bilirubin was not getting into her intestines and instead some had leaked into her bloodstream and was appearing in her urine. The whites of her eyes and skin had become yellow because of the very high level of conjugated bilirubin in her blood.

Now to the gallstones.  In Western countries they are most commonly made of cholesterol.  I have already talked about the importance of cholesterol in keeping cell membranes rigid and non-leaky.  Cholesterol is made in large amounts by the liver and transported to other cells gift-wrapped as LDL cholesterol.  It is also used to make bile salts.  It only needs a minor modification of the basic chemical structure of cholesterol to make bile salts – the main component in bile which makes it work – the main function of bile salts is to help us digest dietary fat.

structure of cholesterol
structure of cholesterol

Bile salts are detergents.  Just like washing-up liquid, they emulsify fats, breaking large fat globules into smaller micelles which do not stick to each other, or anything else. Cholesterol is essentially very insoluble in water, or hydrophobic.  When an organic acid group is added – to make bile salts – it gains a hydrophilic or water-attracting group. This is just like soap – a long lipid chain with an organic acid group at the end.  The hydrophobic lipid part is embedded in the tiny fat globule and the hydrophilic groups stick outside in the watery medium of the intestinal contents.

bile acids are a minor modification of cholesterol - hydrophilic OH  and COOH  groups are added to make it into a detergent
bile acids are a minor modification of cholesterol – hydrophilic OH and COOH groups are added to make it into a detergent

As well as bile salts, the liver secretes phospholipids into bile – principally phosphatidylcholine. This, with cholesterol, is the main stuff cell membranes are made of.  It is also an emulsifying agent, and is used extensively in the food industry to keep fats in suspension.

So, when we eat fat, the small intestine detects it and releases the hormone cholecystokinin (CCK).  This causes the gallbladder to contract to send bile into the duodenum, and also makes the pancreas release enzymes, including lipase.  CCK also has some very interesting effects on our brains, making us feel less hungry, and curiously, opposing the effects of opiate drugs.  The bile and pancreatic secretions are both delivered into the duodenum through the same tube – see diagram. The detergent bile salts, emulsifying phospholipid and pancreatic lipase are designed to work together to digest fat.

the hydrophilic parts of cholesterol are all on one side of the molecule - this allow it to act as a detergent - it works well to disperse fat into small globules so that pancreatic lipase can work on it and break it down to fatty acids and monoglyceride
the hydrophilic parts of cholesterol are all on one side of the molecule – this allow it to act as a detergent – it works well to disperse fat into small globules so that pancreatic lipase can work on it and break it down to fatty acids and monoglyceride

The bile breaks it up into tiny globules and the lipase breaks triglyceride into fatty acids and monoglyceride. These are transported across the intestinal mucosal cell membrane and then the triglyceride is put back together again. Seems a daft system, but it clearly works. The triglyceride is taken away from the intestine not in the portal blood, like most other substances absorbed by the gut, but in the lymphatic system in the form of chylomicrons – small fatty globules covered by a layer of phospholipid. The chylomicrons travel up the lymphatic vessels to emerge into the circulation just below our left clavicle. This means that it avoids passing straight away through the liver. If blood is taken soon after a fatty meal and centrifuged, it will have a milky appearance because of the large amount of chylomicrons.

So why do gallstones form? As well as bile salts, unmodified cholesterol is also secreted into bile. It is only kept soluble by the bile salts and phospholipid – the ratio of cholesterol to bile salts and phospholipid is therefore important. When it goes wrong, cholesterol precipitates out and forms solid stones like those that caused all Sylvia’s recent problems – a bit like when I try to make mayonnaise and it curdles – the fat becomes un-emulsified and separates out.

In some parts of the world, gallstones are made from bile pigment – bilirubin. This can be due to increased red blood cell breakdown resulting from abnormalities such as sickle cell disease, but it’s also common in East Asia, for reasons which are not clear.  A good, comprehensive analysis of why gallstones of all types form can be found at:

http://gastro.ucsd.edu/fellowship/materials/Documents/Gallstones/pathogenesis%20gallstones.pdf

Now the alkaline phosphatase. Bile is alkaline.  Alkaline phosphatase is an enzyme that removes phosphate groups in alkaline conditions.  When bile flow is blocked – by stones, as in Sylvia’s case, or for any other reason – the liver makes much more alkaline phosphatase, some of which appears in the blood.  Bones use a similar alkaline phosphatase to rearrange phosphate groups to make hydroxyapatite – the hard stuff bone is made from.  Most of the alkaline phosphatase normally in our blood is the bone sort.

There is plenty of information about whether alkaline phosphatase is from bone or liver, but very little I can find that suggests why the liver should make this enzyme when the biliary system is blocked.  I think a clue here is that neutrophils also have alkaline phosphatase in their granules, which help kill bacteria.  This is in addition to the myeloperoxidase and esterase mentioned in earlier posts.  Why should a phosphatase be damaging to bacteria?  The answer may well involve techoic acid, an important reinforcing molecule in some gram-positive bacteria.

cartoon of gram positive bacterial cell wall - techoic acid is thought to inhibit the action of lysozyme and protect the peptidoglycan from being broken down - techoic acid contains phosphate groups which may be removed by alkaline phosphatase
cartoon of gram positive bacterial cell wall – techoic acid is thought to inhibit the action of lysozyme and protect the peptidoglycan from being broken down – techoic acid contains phosphate groups which may be removed by alkaline phosphatase

In these microbes, techoic acid strengthens the peptidoglycan cell wall and inhibits the action of lysozyme, yet another enzyme made by neutrophils.  Lysozyme is designed to break some of the sugar-sugar bonds in peptidoglycan – so getting rid of the techoic acid would surely be helpful.  We know that alkaline phosphatase is effective in breaking up techoic acid – see, for instance:

http://jb.asm.org/content/102/3/747.short

Maybe the liver is producing this anti-bacterial enzyme to help prevent infection when the flow of bile slows down.  Unfortunately, this won’t work with gram negative organisms such as E.coli because they don’t make techoic acid – and it was a gram negative bacterium we grew from Sylvia’s blood culture – the germ that was causing her ascending cholangitis.

Soon after she came in, Sylvia had an ERCP – an endoscopic retrograde cholangio-pancreatoscopy.  The endoscopist managed to remove the impacted gallstone from her ampulla of Vater, and she quickly recovered.  A week later she had her gallbladder removed, including the stones, and now is quite well – she can even eat fish and chips.

Now to the food link: mayonnaise.

French mayonnaise - made with olive oil and mustard
French mayonnaise – made with olive oil and mustard

This is an emulsion of lipid, such as olive oil, in an aqueous (watery) medium – vinegar or lemon juice.  The emulsifying agent is raw egg yolk, which – just like bile – contains lots of phospholipid – phophatidylcholine and is also rich in cholesterol.  In France, mustard is also added.  Mustard seed contains mucilage, a gooey stuff that some plants produce made of sugar polymers. (Thanks again to Harold McGee). These also act as emulsifiers and further thicken the mayonnaise, and give it a better taste. When I make cheese sauce, I use a sugar polymer (flour) to achieve a stable emulsion between butter and milk – no doubt the phospholipid in the milk helps too.  It seems likely that in the small intestine, the mucus from the stomach, a similar gloopy sugar polymer, probably has a similar effect to mustard seed mucilage to help with fat emulsification.