Some Leading Questions About Lead

EiU9fU4Z2NV7CzJweYbiNxGnThe ancients were drawn to lead because of its combination of properties: low-melting, very malleable and resistant to corrosion. But it turned out to be very toxic. Although lead’s dark side was revealed a long time ago, lead poisoning is still not a thing of the past. The health of workers and children continue to be seriously compromised as precautions in dealing with lead are ignored domestically in several places and abroad. Hopefully this set of four questions and answers will help the reader become a more vigilant and enlightened citizen with regard to lead.

1. A while ago, one of my summer jobs as a student involved working for a lab in a copper refinery. Why did our blood have to be monitored for lead when lead is not an impurity of copper ore?

Although lead is not an impurity of copper ore, silver, platinum and gold are. After electrolysis causes copper to deposit on the anode, the precious metals precipitate at the bottom of the electrolyte solution. After gold is separated from the other metals, its purity is measured by an ancient but accurate technique know as fire assay. And that’s where lead(Pb) enters the picture. Pb is used in that type of analysis because of its ability to dissolve gold and leave impurities behind. Its melting point is lower so that it can subsequently be separated from the purified gold. The gold sample is weighed before and after the assay, completing the purity-calculation.

Before the 1980s, when precautions were not taken in our fire assaying lab, the technicians developed high levels of lead in their blood. They were given paid leave to recover. When they returned to work, they were provided with a lead-free area in which they kept their non-work clothes apart from the ones worn in the lab . They had to wear respirators during the analyses and ventilation was improved. Routine blood analyses also became the norm for all employees in case they were assigned to work in the fire assay department or in nearby labs.

Unfortunately due to low standards, lead is still a significant occupational hazard in China. As the Chinese authors of this 2010 study point out:

Lead (Pb) and its compounds remain the leading cause of chronic poisoning (51.83%), followed by benzene (13.74%) and trinitrotoluene (TNT) (11.05%); 82.97% of the cases were distributed in medium- and small-scale enterprises. Nowadays, the risk factors of occupational poisoning have been transferred rapidly from developed countries to our country, from urban to rural areas, from developed areas to less developed regions, and from the formal to the informal business sectors.

thecloudmindershd0263.jpg
In the 1969 Star Trek episode The Cloud Miners, the higher class consider themselves superior to the Troglytes who work in mines. With high conventional IQs but low ecological-IQs, the elite didn’t realize that the work environment of their servants exposed them to a poisonous gas that rendered them aggressive and mindless. Undoubtedly, this episode was inspired by the fact that children of poor neighborhoods and certain occupations are more exposed to lead and its serious consequences.

2. Why is lead so toxic?

Lead can damage through a variety of mechanisms, but we’ll look at the two most important ones.

A- Oxidative stress.

Flora_F1
Lead(Pb) increases free radical production(specifically, reactive oxygen species(ROS))  and lowers cells’ capacity to defend against free radicals. Diagram is from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485653/

Organisms either deliberately or inadvertently create uncharged, short-lived molecules with unpaired electrons. These so-called highly reactive free radicals can come in handy, for example, as one of the immune system’s defensive mechanisms. But free radicals can also turn against the host. Lead’s presence causes an overproduction of free radicals while also interfering with our ability to repair the damage from free radicals. (see adjacent diagram)

B- Ionic mechanism. A common charge for the lead ion is +2. Its ionic radius is also similar enough for cells to confuse it for calcium +2. This so-called ionic mechanism is what makes it toxic neurologically. Disguised, lead ion sneaks in across the protective blood-brain-barrier, and accumulates in astrocytes, specialized cells that help keep brain chemistry in balance.  astrocyteThey can get Pb2+ out of the way with the help of binding proteins But the young developing brains of children have immature astrocytes that lack lead-binders. Lead’s unconfronted presence damages the specialized cells, interferes with the formation of the protective myelin sheath, and compromises the development of the blood-brain-barrier.

The ionic mechanism interferes with neurotransmitters, which lowers the intellectual quotients of children who have been exposed to lead. Children, when compared to adults, retain 3 to 6 times as much of the ingested toxin.


3. Why are there no excuses for the existence of lead pipes in our cities?

Considering the deadly nature of lead poison and the fact that so many natural waters dissolve this metal, it is certainly in the cause of safety to avoid, as far as possible, the use of lead pipe for the carrying of water which is to be used for drinking.

Amazingly, the above is from an 1845 report on water supplies for the city of Boston. Despite the warning, cities went ahead and used lead pipes in building water-distribution networks in the late 19th and early 20th centuries. Currently over 100 000 citizens in Montreal  are served by lead pipes.  Only in 2006 did they introduce a 20- year plan to completely remove them. In the meantime city officials advise households in affected neighborhoods with pregnant women and young children to install charcoal filters.

Luckily, leaching of lead is reduced by the introduction of phosphate ion during the water treatment phase. It causes a protective scale to build up inside the lead pipe, keeping most of the lead out of the drinking water. But such a procedure is a temporary, albeit, vital solution. When that chemical treatment has been ignored, tragedies such as the one in Flint, Michigan in 2014-15 have ensued. Meanwhile other American cities with ageing infrastructures continue to violate recommended concentrations of lead in drinking water.


 4. Were lead levels in some Flint, Michigan homes so bad that they could have been detected by a simple wet-chemistry test?

Having often shown students how lead ion’s presence can be revealed by adding iodide ion and creating a startling canary-yellow precipitate, I wondered if an informed citizen of Flint would have been able to see such a product from carrying out the test on their drinking water.

p1060146During the crisis in Flint Michigan, homes had as much as 13 200 ppb (part per billion) or 13.2 mg /L or 13 ppm of lead. The simple iodide test has a   threshold of 20 ppm, so the answer is no; the wet-chemistry test is not sensitive enough, but astonishingly, the concentration of lead in their tap water wasn’t far off from the wet-chemistry’s detection limit.  Instead, atomic absorption was required to detect concentrations in the range of 0.005 mg/L  (= 5 ppb = recommended maximum) to 20 mg/L. It was thanks in part to the inspirational volunteer lab work of the Virginia Tech Research Team that the lead problem in Flint was brought to light.

Other Sources:

Casarett & Doull’s Toxicology: The Basic Science of Poisons. McGraw &Hill

Brent JA. Review of: “Medical Toxicology” Clin Toxicol. 2006;44:355–355.

Bellinger DC. Lead. Pediatrics. 2004;113:1016–1022. [PubMed]——

https://www.canada.ca/content/dam/…vie…/water…/water-corrosion-eau-eng.pdf

http://www.healthycanadians.gc.ca/health-system-systeme-sante/consultations/lead-drinking-water-plomb-eau-potable/document-eng.php#a1

C and Eng Volume 94 Issue 7 | pp. 26-29 | Latest News Web Date: February 11, 2016

The Causes of Heart Disease: Mostly Not Genetic

Some diseases such as chicken pox, TB, maple syrup urine disease and sleeping sickness are rooted in a single cause of a viral, bacterial, genetic and parasitic nature, respectively. But the two most common cardiovascular diseases, atherosclerosis and stroke, which combine to be the number-1 killer of humans worldwide, result from several factors, most of which are not genetic. Yet in too many minds the simplistic view that heart disease results from some pipe-clogging-like agent persists. The excessive focus has shifted from cholesterol to saturated fat and trans fats, while the potential of preventative medicine based on a fuller understanding of the causes of heart disease has been compromised.

1causeDiseases
Four completely different diseases, each brought on by a single cause. Heart diseases , unfortunately, do not fall under such a neat and tidy category.

As early as in the 1970s some medical researchers realised that dietary cholesterol was not cause of atherosclerosis and that the multifactorial nature of the disease was evident. In a 1977 Scientific American article on atherosclerosis, Earl P. Benditt wrote:

Moss and I fed cholesterol and administered estrogen to chickens and examined their vessels with the electron microscope. We found, first of all, that the lesions that cholesterol induced in the chickens did not resemble human atherosclerotic plaques. They appeared to be composed entirely of fat-filled cells derived from blood macrophages; there was no evidence of significant smooth-muscle-cell proliferation. And none of the lesions evolved into the raised plaques characteristic of the human disease.

Poring through recent review articles of the  medical and scientific literature, we observe that the current consensus on atherosclerosis is that’s an inflammatory disease characterised by intense immunological activity.  Moreover, as a November 2016 Nature Reviews Cardiology paper, Nature versus nurture in coronary atherosclerosis, points out:

“Lifestyle factors may powerfully modify risk of coronary artery disease regardless of the patient’s genetic risk profile.”

These findings are consistent with the modified response to injury theory of atherosclerosis. It identifies a number of “insults”, most of which are environmental or linked to lifestyle choices. The following factors trigger the intense immunological activities responsible for heart disease.

(1) Reactive oxygen species from smoking and/or air pollution

(2) High blood concentrations of “low density lipoprotein”(LDL) or “very low density lipoprotein” (VLDL), which contains more triglycerides.

(3) Chronically elevated levels of glucose

(4) Turbulent blood flow from arterial branching

(5) Stress from hypertension or elevated blood pressure

(6) Inherited metabolic defect that raises homocysteine levels, affecting the endothelium of tissue.

Until recently, it was not clear how air pollution led to heart disease. But Mark Miller and his team from Edinburg University and three Dutch institutes wondered whether tiny particles of soot migrate from the lung’s air sacs to the walls of blood vessels of the heart and brain.  Since they used human subjects in the study,  for ethical reasons, the researchers relied on gold, which is biochemically inert. Their patients were asked to inhale soot-sized particles of the precious metal. Within 15 minutes to 24 hours the gold showed up in the blood and urine, and three months later it was still in the body. When mice inhaled gold nanoparticles twice a week for 5 weeks, diseased arteries contained five times as much gold as healthy ones. Three human patients who were already scheduled for heart surgery and who inhaled the fine gold particles ended up concentrating them in their arterial plaques, just like the mice had done.

Pm2_5
Small particles tend to get into the blood stream more easily than larger ones.  From ACS Open access

Upon examining the data, I also found it interesting that small gold particles (less than 10 nm) got into the blood stream far more efficiently than larger ones. This helps supports the current emphasis that environmental scientists place on PM 10 and PM 2.5 pollution, which are not only linked to heart disease but to lung cancer. Also, in the arterial plaque of mice, the gold particles were found within macrophages, suggesting that these may play a role in the translocation of the particles. How the gold is carried around is still not understood, but the authors point out that such knowledge is needed to assess the full impact that inhaled nanoparticles have on health.

macrophage
Inhaled gold particles were found in immune system cells within arterial plaques of mice. From ACS open access

Sources:

The Promise of a Non-addictive Opioid

As far back as the late 1800s heroin was once hailed with the promise of being an abuse-free opiate. Although dozens of other opiates have been created, no one in the past 12o years has succeeded in creating a side-effect-free and non-addictive opioid. But by using a new approach, there is a chance that a German team led  by the anesthesiology and professor Dr. Christoph Stein has succeeded. Before delving into the specifics and exploring the chemistry, let’s provide some background information.

What Are Opioids?

An opioid can be any of these compounds: (1) it can be part of the group of substances directly made by, or derived from, the opium poppy; (2) it can be one of the human-brain-produced peptides that influence a variety of  behaviours including attachment, stress, food-intake and pain response; or (3) it can be a synthetic substance that binds to the same receptors as the previous two types. The receptors known so far include μ (mu—the m coming from “morphine”), δ (delta) and κ (kappa).

Opioids are used to treat pain brought on by terminal or serious illnesses, such as cancer. Some medical practitioners also turn to opioids for chronic pain, common among those with forms of arthritis, back injuries, torn muscles, damaged nerves and fibromyalgia. A case of kidneys stones, nature’s way of making men experience something almost as painful as childbirth, sure made me appreciate a dose of morphine. Since that experience, I have not needed a strong opiate.

Others are not as lucky and need more intense compound-assisted pain management, especially if other opioids have become ineffective. Fentanyl (trade names: Actiq®, Duragesic®, and Sublimaze®) is a quick-acting synthetic opioid with 50-100 times the strength of morphine. But transdermal patches of fentanyl are prescribed only when at least a week of tolerance towards opiates has already been established. That’s because with all its delivery-forms, the use of fentanyl can be very risky. Opioid receptors are all over the body, brain included, and fentanly will not only cause dopamine levels to spike, but it could also bind to brain receptors that control the breathing rate. Respiratory depression can be fatal, and the online availability of illicit fentanyl sold through decoy packages has led to an epidemic rise in fentanyl-related deaths in Canada, Estonia and the United States.

WEB_urinestrips
The first pouch on the right hides a few grains of fentanyl (enough to kill). The package was mailed from China pretending to be a set of urine-testing strips. From the Globe and Mail

Other undesirable reactions from binding to μ receptors include reduced gastrointestinal nausea, vomiting, and the accompanied euphoria from the ensuing dopamine-spike, which ties into addiction.

How A Safer Alternative Was Found

The researchers started by focusing on the role of MOR (μ opiate receptors) in injured tissues as opposed to the brain. They did this for two reasons (1) Painful conditions such as inflammation or trauma are often associated with a lower pH(acidification) in localized tissue. (2) Only the protonated form of opiates like fentanyl can bond to MOR receptors. To be protonated simply means that an H+ ion has been attached to a basic group, in this case the nitrogen of a heterocyclic ring called piperidine . Then through hydrogen bonding, that proton attracts the oxygen of a carboxylate group of an aspartic acid molecule on the receptor.

fentanylCapture
The protonated fentanyl shown bonded to an aspartic acid residue (Asp 147) of a μ opiate receptor. Modified from author’s paper in Science.

Fentanyl and other opiates have substantial fractions of their molecules protonated at pHs of about 5 to 7, where the source of pain lies.  But the problem is that opiates are also highly protonated at pH 7.4 , the typical pH of the brain and small intestines where you don’t want the opiates to be activated.

We will represent the protonated form of fentanyl as HNFen+ and the uncharged basic counterpart as NFen:

HNFen+ = H+ + NFen

What the researchers aimed to do was to attach a fluorine group to key positions of the fentanyl molecule to increase its acidity without altering the stereochemistry or functional groups of the rest of the molecule. After some computer modelling they suggested the synthesis of NFEPP:

Nfepp
The protonated form of NFEPP, which has been shown to be a non addictive opioid still capable of acting as an analgesic. This form becomes less important above a pH equal to its pKa of 6.8
fentanyl
The protonated form of fentanyl found in the commercial citrate. It’s also the predominant form below pH = pKa = 8.4, so in other words in any human tissue containing opioid receptors.

Even at the beta position (on the second adjacent carbon attached to nitrogen), fluorine’s electron-withdrawing presence decreases a nitrogen’s ability  to reattach itself to H+. This happens because nitrogen uses its electron pair to act as a base and bond to H+. If the reverse reaction is less likely to proceed, the acidic forward reaction is favoured. That is a good thing for the drug designers because they did not want the protonated form to dominate in certain tissues where it could lead to addiction and respiratory arrest.

To understand why a more acidic molecule will be adequately protonated in the proton-rich environment —where the pain is—and why there will be far less protonated than fentanyl at pH=7.4, let’s explore some analytical chemistry.

For weak acid systems like

HNFen+ = H+ + NFen , we cannot make the assumption that the H+ concentration will equal that of NFen. To estimate the relative concentration of the protonated fentanyl  we have to rely on the mass balance approach where we compare HNFen+ ‘s concentration, [HNFen+ ], to that of the total of  non H+ species.

We let X = [HNFen+ ]+ [NFen] = sum of concentration of non-H+ species.

so [NFen] = X – [HNFen+ ]

since its acid dissociation constant, Ka = [H+][NFen]/[HNFen+ ], by susbtituting for [NFen] we obtain:

Ka = [H+](X – [HNFen+] )/[HNFen+].  By then solving for [HNFen+ ], we obtain:

[HNFen+] =  [H+]X / ( Ka + [H+] )

Finally by rearranging the expression, we get the fraction of protonated species as:

[HNFen+]/X = [H+]/ (Ka + [H+] )

The expression suggests that the fraction of the protonated species is pH-dependent and is inversely proportional to the strength of the acid (measured by its Ka). If [H+] = Ka or if pKa = pH , we will get a mole fraction of exactly of 0.5. This implies that since NFEPP’s pKa is lower (6.8) than fentanyl’s, then the concentration of the protonated form will be the minority-species at a pH that’s lower than the crossover point for fentanyl. The latter has a lower Ka and hence higher pKa of 8.4. To better reveal the relationship, I plotted the fraction [HNFen+]/X versus the pH for both fentanyl and for NFEPP, which due to its fluorine has a higher Ka.

A-pHplot
Notice how the fraction of the protonated form of NFEPP drops dramatically to about 10% at pH= 7.4 whereas that of fentanyl is still above 90%. Meanwhile NFEPP still acts as an analgesic because at pHs of 6.8 to 5.2, the fraction of the protonated form that’s needed to bond to μ receptors in inflammation areas is in the 52 to 95% range, respectively.

But does all this theory work in a biological environment? In the authors’ experiments, NFEPP produced analgesia in rats who had different types of inflammatory pain. And equally important the pain relief was not accompanied by typical opiate side effects such as sedation, decreased breathing, constipation or the desire to seek more pain-killer. In 4 to 5 years, the time it will take to refine the synthesis and test the product in humans, it will be interesting to see if the usual unforeseen consequences will be minor. Of course NFEPP ‘s inability to produce euphoria will not displace dangerous opiates from the black market. But it will help alleviate the problem by putting nonaddictive drugs into circulation while hopefully making the legitimate production and prescription of fentanyl and other opiates obsolete.

Sources:

A nontoxic pain killer designed by modeling of pathological receptor conformations http://science.sciencemag.org/content/355/6328/966

Fluorine in Medicinal Chemistry ChemBioChem 2004, 5, 637 ± 643 https://ccehub.org/resources/611/download/637_ftp.pdf

http://consumerhealthchoices.org/wp-content/uploads/2012/08/BBD-Opioids-Full.pdf

A brief history of opiates, opioid peptides, and opioid receptors Michael J. Brownstein Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20892  http://www.pnas.org/content/90/12/5391.short

http://www.sciencedirect.com/science/article/pii/S0304395904004476

https://www.drugabuse.gov/publications/drugfacts/fentanyl

http://www.theglobeandmail.com/news/investigations/a-killer-high-how-canada-got-addicted-tofentanyl/article29570025/

http://www.theglobeandmail.com/news/national/estonias-cautionary-fentanyl-tale-for-canada/article30512811/

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