Heavy Metal
Chasing the elusive cause of one dog's failing liver
Nature rarer uses yellow
Than another hue;
Saves she all of that for sunsets, —
Prodigal of blue,
Spending scarlet like a woman,
Yellow she affords
Only scantly and selectly,
Like a lover's words.
— Emily Dickinson,
“Nature Poem XXXI”
Nature Rarely Uses Yellow
The first thing they noticed was his gums had turned the color of mustard. Zip’s owners were understandably concerned and rushed him to their family vet to figure out what was wrong with their five year old Australian cattle dog. As soon as the doctor walked in the room, she saw the whites of his eyes had yellowed and knew what was wrong—this was jaundice, an uncommon exam finding, and always a sign of serious disease. Jaundice occurs when a waste product derived from hemoglobin called bilirubin builds up in the blood and discolors tissues.
Preliminary bloodwork showed Zip had severe elevations in an alphabet soup of different liver enzymes like ALT, AST, and ALP. His clotting times were off the chart. Unsurprisingly, he had severely increased bilirubin, as well as evidence that multiple compounds synthesized by the liver (like albumin and urea) were not being made in sufficient quantities. His urine, too, was loaded with bilirubin, turning it a nauseating cloudy yellow-orange, like Tang.
Zip’s liver was shutting down. The question was why? To figure that out, their vet sent them on their way to the teaching hospital at Auburn University.
The Auburn Critical Care and Internal Medicine services immediately got to work running a battery of tests, casting a wide net to investigate what was killing Zip’s liver. Leptospirosis? Negative. Toxins? No known exposure, and didn’t fit the clinical picture. Chest x-rays were normal, but an abdominal ultrasound found the liver was mottled like Swiss cheese. This is a pattern that can often be seen with cancer. To rule this possibility out, they carefully inserted a five-inch spinal needle into the diseased organ to collect cells for cytology.
This is where I come into the story. I was on duty in the clinical pathology lab that day and received Zip’s aspirates. As soon as I started looking at the slides under the microscope it was clear this wasn’t a typical liver case. First of all, groups of hepatocytes (liver cells) were disorganized and appeared atypical. Not quite cancerous, but simply…off. Next, there was marked inflammation as well as fibrosis (which we often can’t see on cytology). The weirdest thing we observed was that many of the liver cells were loaded with dark blue-black pigment…
What could that be? It wasn’t the right shade of green for bile or lipofuscin (a normal “wear and tear” pigment). The top culprits were toxic heavy metals, mainly copper or iron. We needed a full biopsy to investigate further.
The pathologist who received Zip’s biopsies had decades of experience under his belt, and was an expert canine in liver disease. His diagnosis was severe chronic hepatitis. There was necrosis and significant scarring that suggested end-stage disease (cirrhosis). He also noted that the tissue was packed with the most iron he had ever seen in his career. We submitted additional blood tests to an outside lab to evaluate his whole body iron levels and they were stratospheric.
Zip was on a laundry list of medications at this point, including steroids to try and reduce the inflammation, antibiotics to clear any lurking infection, Vitamin K to prevent bleeding to death—clotting factors are made in the liver—and several liver-protecting drugs like Ursodiol and Denamarin. His liver enzymes continued to climb despite these treatments. Clinically, he was still highlighter yellow and felt like crap. Nothing seemed to be working.
The odds were not in his favor. While dogs diagnosed with chronic hepatitis early in the course of their illness can live for over a year, those as advanced as Zip tended to fare poorly:
“In the 10 studies with survival data (n = 364 dogs), mean survival time was 561 ± 268 days. In dogs with biopsy-proven cirrhosis, survival was considerably shorter, 23 ± 23 days (n = 39).” — ACVIM Consensus Statement on chronic hepatitis in dogs
Zip was a friendly dog who was a pleasure to visit in the wards, and I had become invested in his case. We had many discussions over books and studies with his care team during those precarious weeks, racking our brains for a breakthrough. I could not shake the notion that the iron clogging his liver was the key. Iron overload— hemochromatosis—is a fairly common cause of liver failure in people as well as zoo and wildlife species in captivity, like black rhinos. However, hereditary hemochromatosis had never been diagnosed in dogs.
Running out of time and ideas, we discussed the possibility of iron overload with Zip’s owners. Nutritional changes were low risk, so they agreed to switch to an iron-restricted diet. The next intervention was more of a stretch: because red blood cells are loaded with iron-rich hemoglobin, one of the mainstays of hemochromatosis management in people is phlebotomy, or removal of blood. While no one was excited about embarking on this unusual treatment, we decided it was his last shot. That summer, we began harvesting a unit of blood every 1-3 weeks and anxiously monitoring him to see if it was helping or hurting.
Draining Discolored Blood
Iron is essential for carrying oxygen through the blood as well as catalyzing numerous chemical reactions in the body. However, as with many things in life, too much of a good thing can become harmful. Even mildly increased iron levels are toxic and cause tissue damage. Left untreated, it can cause hepatitis and cirrhosis, and increases the risk of liver cancer. The other manifestations of hemochromatosis are protean and range from diabetes and sex hormone problems, to bone marrow failure, bronzed skin, brittle nails and hair loss, and severe arthritis. The word hemochromatosis literally means a “disease of colored blood” in Greek, poetically named for the build-up of the dark metal in skin and other organs.
Hemochromatosis was first described by a French physician in 1865, but medicine had a murky understanding of the disease until molecular techniques advanced in the 1970s-1990s and allowed us to solve the puzzle. The hereditary form in people arises from defects in one of at least five genes that control iron absorption and metabolism, leading to uncontrolled build-up of the heavy metal. It is one of the most common genetic diseases in the world, affecting approximately 1 in 200 people of northern European descent, and up to 1/3 of select ethnicities are carriers. In fact, I inherited one copy of the less severe mutation, but fortunately do not have the condition.
The prevalence and severity is higher in men than women (menstruation is likely protective by depleting iron) and alcohol consumption is one of the biggest risk factors. The main approach for both prevention and treatment of hemochromatosis is phlebotomy, essentially a modern version of the folk remedy bloodletting. This is usually arranged through periodic donation of blood. Those bags of blood used to be discarded as medical waste, but recently the Red Cross and other blood banks began allowing them to be used. Other treatments include diet modification and iron chelation, but they are less effective.
Bloodletting has a checkered history in medicine. It has been practiced for thousands of years by the ancient Egyptians and Greeks, and surprisingly persisted in Europe well into the 1800s (the red and white striped barber poles we see today trace their origins to the practice of barber-surgeons slicing open veins). The Greek physician Galen taught that all forms of disease emanated from an imbalance in four bodily fluids, or humors: blood, phlegm, yellow bile, and black bile. Many modern phrases stem from this concept. Depression, or melancholy, was believed to result from an excess of black bile (“melan” = black, “chole” = bile), while someone with an excess of yellow bile was said to have an angry “bilious” or “choleric” temperament. Blood was considered the most important humor and the easiest to access, so bloodletting—either through leeches, venesection, or less savory means—was prescribed as a cure-all for virtually any condition.
With medical advances in the 20th century, phlebotomy fell out of favor as pseudoscientific nonsense. However, they say all things come back in style if you wait long enough; it would later return as a legitimate treatment for several blood disorders, mainly hemochromatosis and polycythemia vera (overproduction of red blood cells). Time really is a flat circle.
Bloodletting—either through leeches, venesection, or less savory means—was prescribed as a cure-all for virtually any condition
Fortunately for us, Zip responded to phlebotomies extremely well. He did not mind coming in frequently and became a minor celebrity in the hospital. His owners were happy to report his symptoms improved and his lab abnormalities normalized. In fact, a repeat liver biopsy six months later showed that his liver was less inflamed, with reversal of iron accumulation and fibrosis, which is almost unheard of in chronic hepatitis. What’s more, he had been weaned off medications during this time.
Zip would occasionally develop flare-ups where he would feel lousy, stop eating, and have trouble walking due to joint pain. These were usually preceded by increases in his iron levels, and he would improve after a phlebotomy. Everyone involved with the case was amazed with him not only surviving—first six months, then a year, then 18 months—but thriving.
White Whale
Around the time we were scratching our heads for any ideas to help Zip, I reached out to our professional society email list-serv to get some open source feedback. Several prominent hematologists and liver gurus chimed in with their opinions. The general consensus was…maaaaaaaaaaybe. There were some aspects of the case that fit, while others didn’t. They agreed it was suspicious, but the only cases of hemochromatosis that have ever been diagnosed were secondary to outliers that didn’t apply, like chronic blood transfusions or hemolytic anemia. One eminent hematologist described hereditary hemochromatosis in dogs as his “white whale,” something he had suspected in numerous cases over his career, but was never able to prove.
Zip was doing so well on phlebotomies I was convinced that he had to have some defect in his iron pathways. All of the pieces fit, from his lab work, to his amazing response to therapy, living far beyond our expectations, to the fact that some of his littermates had unexplained elevations in liver enzymes, too (we were unfortunately never able to get samples from them to test).
I set to work examining the molecular pathways to try and crack the secrets of his failing liver. First, we ran PCR and sequenced the HFE gene, the main culprit in 95% of people with hemochromatosis. There is less available information about the normal canine genome than the human genome, but from what we could tell, there were no obvious mutations in HFE. Another experiment showed that his liver was overexpressing hepcidin, the master controller of iron absorption. However, each gene in that pathway we tested was (frustratingly) normal.
We had hit dead end after dead end.
Doubt set in. While iron overload is not currently recognized as a cause of chronic hepatitis in dogs, we do know that iron can increase secondary to liver disease, creating a chicken-and-egg problem. Inflammation can mess with iron levels. So can some medications. The waters were muddy, to say the least.
Was my theory just confirmation bias, or a burning desire to be the first to document a new disease? Perhaps I had gotten too close to the case, and simply wanted to believe our unconventional management was helping. No doctor wants to think that they ordered an ineffective speculative treatment. Maybe he was just an outlier chronic hepatitis patient beating the odds.
We had hit dead end after dead end.
Over the following years, my curiosity turned to obsession, and I too felt like Captain Ahab chasing his elusive white whale. I would be swimming laps at the gym and think of a new pathway to test. Then the results would be negative or inconclusive. While bored in a committee meeting, I’d find myself reading new studies on hemochromatosis. I’m sure my wife was sick of hearing about about iron 24/7. Every new explanation had a counter-argument, and a counter-counter argument, on and on through infinite recursions. Answers always seemed just out of reach. The ambiguity and uncertainty gnawed at me.
Epilogue
Every story ends, eventually. More than two years after Zip first walked into Auburn, his owners elected euthanasia. He had been having progressively more flare-ups and stopped responding to treatment, and his owners decided it was time.
On autopsy, we did not find evidence of iron accumulation in his tissues. However, he had recently received phlebotomy before he died, which will deplete those stores. The final report diagnosed chronic liver disease, and not much else. When I read it my heart sank. However, as others have said before, “autopsies give us the facts, but not the truth.”
Zip lives on through teaching and research. Countless students and veterinarians have heard his story in my lectures about iron biology and testing. His case triggered my research interest in iron biomarkers (and hematology more broadly), and we’ve published several studies on the iron response of dogs to trauma and inflammation.
Maybe it’s madness, but I still haven’t given up on the possibility that there is some mutation in Zip’s DNA, just waiting for us to discover. Even in human medicine, which is decades ahead of veterinary medicine in understanding this disease, novel forms of hemochromatosis with unique gene defects are being discovered every year. I plan to submit the DNA we have in the freezer for whole genome sequencing. Maybe someday we’ll have an answer, and closure.
This is from someone (me,) who knows no medicine at all! So don't fall over laughing, but could Zip's hepatic problems have their origin in Zip:s brain? Do brain and liver communicate? Could something have gone awry "upstairs"? Previous viral infection there?
My day job is a Medical Laboratory Scientist at a major hospital! This story was an amazing read!
With humans, patients with HH are able to donate blood, would you be able to use the blood collected from the Aussie in other patients?