Nitric Oxide Theory of Aging

Interesting information on NO is oral cavity and gastric mucosa, and the role of bacteria in digestion of NO - which Andrea does a great job explaining as well. 

NO does appear to have beneficial qualities in allowing blood vessel dilation and increase blood flow to occur at needed times. On the other hand, these effects seem to be needed at times in cases due to disruption in normal function - such as by viral or endotoxin exposure (1,2). The NO theory of aging  suggests that repeated infections create progressive oxidative damage leading to coronary disease and neurodegenerative conditions (1,2). The principal mechanism tested is when bacterial lipopolysaccharides (LPS) injected, NO Synthetase is induced (via an IL-1) mediator within 2-4 hours (2). The blood brain barrier does not appear to keep LPS out of the central nervous system (3), and an intense NO response is seen in the brain (1,2). The effect of NO is to inactivate enzymes which leads to cell death of invading virus as well as host cells (1). Antioxidant vitamin C and E appear to be beneficial in attenuating the NO oxidant effect (1,2). 

Sources:

1/ McCann S et al. The nitric oxide theory of aging revisited. Ann N Y Acad Sci. 2005 Dec;1057:64-84.

2/McCann S et al. The nitric oxide hypothesis of aging. Exp Gerontol. 1998 Nov-Dec;33(7-8):813-26.

3/Wei-Ye L et al. Increasing the Permeability of the Blood–brain Barrier in Three Different Models in vivo. CNS Neuroscience & Therapeutics. 21 (2015) 568-574.

Vascular Risk Assessment

Below you will find case histories on 2 individuals (these are real clients that I've seen in the past
couple of years). Based on the information provided, how do you assess the risk for cardiovascular
disease in each individual? Who do you believe has the greater cardiovascular risk and why?
What further assessments and recommendations might be appropriate for each?

Case # 1 RM is a 50-year-old male who is 6 feet tall and weighs 305 pounds. He has marked
central obesity and a sedentary lifestyle. His diet is primarily "meat and potatoes" with a large
amount of fast food. Lab results as follows:
glucose 88 mg/dl
cholesterol 195 mg/dl
triglycerides 87 mg/dl
HDL 59 mg/dl
LDL 119 mg/dl
Blood pressure 125/85

Case #2 LC is a 49-year-old female who is 5 feet tall and weighs 100 pounds. She runs 3 to 4
miles daily and tries to eat a "healthy" diet, including many vegetables, beans, fish, and lean meat.
Lab results as follows:
glucose 93 mg/dl
cholesterol 214 mg/dl
triglycerides 51 mg/dl
HDL 77 mg/dl
LDL 127 mg/dl
Blood pressure 115/70
Both individuals are nonsmokers.
______________

I reviewed the risk as calculated by the Framingham Coronary Prediction algorithm and
summarized here:

Case 1 has the higher calculated risk before taking into account a very high BMI, as well as a
likely hip-waist ratio that also makes him at risk. I agree with Miranda's observation about Case
2 where the intake of calories may not match the physical demands she is placing on her body
which can lead to hypothyroid and high TC. I know several long distance runners who have run
into health issues with cardiac arrhythmia related to the bradycardia. Case 1 to lower risk needs
to be more active and be more particular about diet choices. One could argue that hypothyroid
concerns are present in Case 1 exercise/diet choices showing up with borderline elevated TC.
Fast foods can be laden with rancid (old) vegetable oils which can suppress thyroid by multiple
mechanisms. (1) Additionally, overeating muscle meats tends to lead to imbalance between
more phosphate and less calcium which can activate parathyroid hormone. (2)

Sources:
(1) Tom Brimeyer. The Worst Food for Your Thyroid. 4/25/18.
http://www.forefronthealth.com/worst-food-thyroid/ (http://www.forefronthealth.com/worstfood-
thyroid/)
(2) Kemi, V.E., Kärkkäinen, M.U.M., Rita, H.J., Laaksonen, M.M.L., Outila, T.A., Lamberg-
Allardt, C.J.E., 2010. Low calcium:phosphorus ratio in habitual diets affects serum parathyroid
hormone concentration and calcium metabolism in healthy women with adequate calcium
intake. British Journal of Nutrition 103, 561. https://doi.org/10.1017/S0007114509992121
(https://doi.org/10.1017/S0007114509992121)

The standard lipoprotein profile

Discuss the individual tests performed in a standard lipoprotein profile. 

A standard serum “lipid panel” includes: total cholesterol, High-density lipoprotein cholesterol (HDLC), Low-density lipoprotein cholesterol (LDL-C), Triglycerides.

Cholesterol is a steroid. A steroid is a 4-ringed carbon organic compound that varies depending on
the attached functional group and by the oxidation state of the rings. Steroids are found as
stabilizing structures in cells, cholesterol, or as hormones (e.g., estradiol, testosterone,
progesterone). Cholesterol can be synthesized by all animal cells for cell structure integrity (93%)
and circulating in blood (7%) as a precursor for steroid hormones, bile acid and vitamin D.
Cholesterol circulates in blood as part of a lipoprotein complex which contains a protein,
cholesterol, triglyceride, and phospholipid molecule. The lipoprotein complex can be classified by
density into HDL, LDL, and very-low density lipoproteins (VLDL).

Triglycerides (TG) are the main components of body and vegetable fat and are made up of a
glycerol and 3 fatty acids (thus TRI-fatty-acid + Glycerol shortens to Triglycerides). There are
saturated and unsaturated types of TG. Saturated TG are saturated with hydrogen, which means
all available places on the carbon structures are occupied. Unsaturated TG have double bonds
between carbon atoms. Functionally we see saturated TG solid a room temperature (e.g. Butter)
and we see unsaturated TG liquid at room temperature (e.g., vegetable oil). Since humans live at
~98 degrees F, saturated TG can be used as they are liquid at this higher temperature. Fish
however, or plants that grow in northern latitudes need liquid fats at colder temperatures and so we
find mostly unsaturated TG in these organisms. With more double bonds, unsaturated TG are more
prone to oxidation – “oil oxidation is an undesirable series of chemical reactions involving oxygen
that degrades the quality of an oil” (4).

Lipoproteins carry hydrophobic molecules in serum and consist of a single layered membrane of
phospholipids with the hydrophilic portions (the phosphate head) facing outwards and the
hydrophobic portion (the fatty acid tails) facing inward. Apolipoproteins are embedded in this
membrane of phospholipids to stabilize the structure and provide different functions. The various
lipoproteins consist of plasma lipoprotein particles classified as HDL, LDL, IDl, VLDL, ULDL
(chylomicrons). Other lipoproteins include the transmembrane proteins of mitochondria.
HDL is often called "good cholesterol" because it removes excess cholesterol and carries it to the
liver for removal. LDL is often called "bad cholesterol" because it deposits excess cholesterol in
walls of blood vessels, which can contribute to atherosclerosis.

LDL particles package fatty acids for delivery around body via serum. Each LDL particle contains a
single apolipoprotein B-100 (Apo B 100) molecule, and contains a hydrophobic core containing
cholesterol molecules and TG. LDL particles are ~25 nm in size and have a 3 million Dalton mass.
LDL size can vary substantially and with new technology (NMR spectroscopy), much smaller dense
LDL particles identified. More small dense LDL particles appears to equate to higher risks for
atherosclerosis than more larger and less dense LDL forms, possibly due to small forms able to
penetrate vascular endothelium more readily. LDL reported on blood tests (LDL-C) is based on a
calculation (the Friedewald equation) in which Total Cholesterol less HDL and TG gives an estimate
of LDL.

From the National Cholesterol Education Program, 2001, elevate risk of heart disease occurs when
TC more than 240 mg/dL. Likewise, LDL levels exceeding 100 have higher risks, especially high
over 160 mg/dL. Higher HDL in this scheme confers less risk at more than 60 mg/dL.
Growing evidence indicates that LDL levels may not correlate so strongly with heart disease
however. In a 1997 prospective Quebec Cardiovascular Study, 2013 healthy men were followed,
and it was found that of the 114 that developed ischemic heart disease that total LDL levels had
virtually no impact, although small LDL particles were associated with increased risk. (3)
Many lipid studies seem to be association studies and as such difficult to prove causation. Other
association studies show that Bacteroidetes bacteria are associated with atherosclerosis, so
perhaps diet that limits endotoxin production is of primary importance in preventing ischemic heart
disease. (4) The endotoxin theory of atherosclerosis has been described. (5,6) Additionally, nitric
oxide, often said to be a beneficial compound, may not be benign and seems to be induced by
endotoxin.

Sources:
1/Nutritional Assessment

2/Moseby’s

3/Enig, Mary. Know Your Fats

4/Oxidation of food grade oils – www.plantandfood.com (http://www.plantandfood.com) . Good
summary on oxidation.

3/Lamarche, B., Tchernof, A., Moorjani, S., Cantin, B., Dagenais, G.R., Lupien, P.J., Despres, J.-P.,
1997. Small, Dense Low-Density Lipoprotein Particles as a Predictor of the Risk of Ischemic Heart
Disease in Men: Prospective Results From the Quebec Cardiovascular Study. Circulation 95, 69–
75. https://doi.org/10.1161/01.CIR.95.1.69 (https://doi.org/10.1161/01.CIR.95.1.69)

4/Nemati, R., Dietz, C., Anstadt, E.J., Cervantes, J., Liu, Y., Dewhirst, F.E., Clark, R.B., Finegold,
S., Gallagher, J.J., Smith, M.B., Yao, X., Nichols, F.C., 2017. Deposition and hydrolysis of serine
dipeptide lipids of Bacteroidetes bacteria in human arteries: relationship to atherosclerosis. Journal
of Lipid Research 58, 1999–2007. https://doi.org/10.1194/jlr.M077792
(https://doi.org/10.1194/jlr.M077792)

5/Got a Bad Ticker? Bacterial Fats Could be the Blame. GEN News Highlights. 11/2/207.
https://www.genengnews.com/gen-news-highlights/got-a-bad-ticker-bacterial-fats-could-beto-
blame/81255124 (https://www.genengnews.com/gen-news-highlights/got-a-bad-ticker-bacterialfats-
could-be-to-blame/81255124)

6/Anikhovskaia, I.A., Kubatiev, A.A., Iakovlev, M.I., 2015. [Endotoxin theory of atherosclerosis].
Fiziol Cheloveka 41, 106–116.

7/Konev, I.V., Lazebnik, L.B., 2011. [Endotoxin (LPS) in the pathogenesis of atherosclerosis]. Eksp
Klin Gastroenterol 15–26.

8/McCann, S.M., Licinio, J., Wong, M.L., Yu, W.H., Karanth, S., Rettorri, V., 1998. The nitric oxide
hypothesis of aging. Exp. Gerontol. 33, 813–826.

9/McCANN, S.M., 2005. The Nitric Oxide Theory of Aging Revisited. Annals of the New York
Academy of Sciences 1057, 64–84. https://doi.org/10.1196/annals.1356.064
(https://doi.org/10.1196/annals.1356.064)
(http

Assessing vitamin C status

Discuss some of the biochemical procedures for assessing vitamin C status. Which are better measurements of dietary intake? Which are better measurements of cellular stores? 

Vitamin C has been postulated to have an important role in reduction in oxidative stress and prevention of cancer and heart disease, and in boosting the immune system (1). Vitamin C deficiency, as is well known, causes scurvy which is typically seen after 1 month of dietary deficiency largely due to impaired collagen biosynthesis (1).

The USDA Dietary Reference Intakes for Vitamin C indicate that the Recommended Daily Allowance for males 19+ years old is 90 mg and females of the same age range is 75 ug/d. Of note, for smokers, there is an additional 35 mg/day recommended. Food sources of vitamin C include fruits and vegetables, especially citrus, tomatoes, potatoes, red and green peppers, kiwifruit, broccoli, strawberries, Brussels sprouts, and cantaloupe. According to the 2001–2002 National Health and Nutrition Examination Survey (NHANES), mean intakes of vitamin C are 105.2 mg/day for adult males and 83.6 mg/day for adult females, meeting the currently established RDA for most nonsmoking adults (1).

Vitamin C exists in the body as (1) Ascorbic Acid, (2) Ascorbate, and (3) Dehydroascorbic acid (3).

Under neutral serum pH, Vitamin C exists predominantly as Ascorbate. As cells absorb Ascorbate, it is oxidized and, in the cell, exists as in an OXIDIZED state as Dehydroascorbic Acid. So you see a flow of electrons from REDUCED state in Ascorbic Acid to OXIDIZED state inside the cell. The OXIDIZED form is then hydrated to Diketogluconic Acid which is excreted in urine (3).

As such it follows that measuring Vitamin C level can come from any of the 3 pools of vitamin C in the body: (i) Urine, (ii) Plasma, (iii) and Tissues, including leukocytes and platelets.

In the Plasma, the most sensitive and selective method for measuring vitamin C uses high-pressure liquid chromatography (HPLC) coupled to an electrochemical (EC) detector. This can be problematic as vitamin C is unstable and requires extra steps be taken including continuous storage at -80^o C. Total plasma vitamin C is measured as ascorbic acid is easily oxidize to dehydroascorbic acid and indeed from the serum, dehydroascorbic acid may be completely produced after the sample is taken and transported to the lab. An UV HPLC technique has been described which requires minimal transport and costs relative to the typical expensive HPLC (3).

In the tissues, vitamin C can also be measured in the leukocytes or platelets and may be more accurate. The levels in urine and plasma vary quickly with intake, vitamin C from tissue may best measure body stores. Studies suggest three different normal reference ranges in tissue, from 1500 to 5,000 mg (20, 22, 32 mg per kg of body weight).  The half-life of vitamin C in tissues varies depending on the literature data, from 16 to 20 days. Assuming a tissue level of 5000 mg, a lack of vitamin C in the diet for 16 days would reduce the tissue store to about 2500 mg; in 32 days it would be about 1250 mg; in 44 days it would be 625 mg and in 64 days it would be about 313 mg and clinical signs of scurvy should start to develop (2).

As many recognize from supplementing with vitamin C, a significant amount appears in urine soon after ingesting. The serum appears to have a saturation point for vitamin C and excess of this water-soluble vitamin excreted by kidney. The renal threshold of plasma vitamin C is ~1.4 mg/dL and further intake results in a rapid urine clearance. Vitamin C can be excreted in the urine unchanged as ascorbic acid, as dehydroascorbic acid, 2,3-diketogluconic acid and oxalic acid (2).

An at home method has been produced to assess urinary Vitamin C levels: Vita-Chek-C (http://store.riordanclinic.org/product/VPLCStrips.html). The VitaCheck-C color chart reflects levels of vitamin C of 0, 5, 10, 20, 40, 50, and 100 mg/dL.

In a study using the Vita-Check-C and Plasma levels, these were the results (2):

Note that many with Plasma levels below the renal threshold of 1.4 still showed significant amount in urine. Therefore this test is a screen and generally over 40 is considered “normal” without clinical suspicion of deficiency (2).

1 / NIH Vitamin A Fact Sheet for Health Professionals: https://www.nal.usda.gov/fnic/ascorbic-acid-vitamin-c

2 / Jackson, et al. Screening for Vitamin C in the Urine:Is it Clinically Significant?. Journal of Orthomolecular Medicine Vol. 20, No. 4, 2005

3 / Robitaille et al. A simple method for plasma total vitamin C analysis suitable for routine clinical laboratory use. Nutr J. 2016; 15: 40.

Alzheimers and PUFA

The potential nutritional strategies to prevent or treat Alzheimer’s perhaps via the ApoE gene/protein is certainly an interest of mine. One concern I have reading through the comments regards recommendations for the intake of fat – namely that saturated fats should be avoided and PUFA should be preferred. At risk of straying too far off mainstream, my sense is that the unsaturated fats, given the higher likelihood to be oxidized than saturated fats, can create some of the situations we are seeing with Alzheimer’s pathology. From this article (1):

“ROS (reactive oxygen species) and RNS (reactive nitrogen species) can substantially disrupt physiological processes through participation in reduction–oxidation (redox) reactions involving the loss (oxidation) or addition of electrons (reduction). Polyunsaturated fatty acids (PUFAs), which are characterized by conjugated double carbon bonds that can act as a source of electrons, are particularly susceptible to oxidative damage. Cell membranes are rich in PUFAs, and free radical damage to PUFAs can reduce the integrity of the membrane.”

In this summary piece on AD (2), these Harvard researchers state that AD is caused by mitochondrial dysregulation due to oxidative damage. For cellular structures made up of PUFA, the damage, it seems to me, will be much greater than with saturated fats which are more resistant to oxidation. (3)  There are not many well designed studies teasing out this issue/

(1)     Da Costa, et al. Genetic Determinants of Dietary Antioxidant Status. Progress in Molecular Biology and Translational Science Volume 108, 2012, Pages 179-200

(2)    https://news.harvard.edu/gazette/story/2015/02/a-new-understanding-of-alzheimers/

(3)    Br J Nutr. 1987 May;57(3):383-93. Differential oxidation of saturated and unsaturated fatty acids in vivo in the rat.

Vitamin C Overview

Vitamin C (or L-ascorbic acid) has been postulated to have an important role in reduction in oxidative stress and prevention of cancer and heart disease, and in boosting the immune system (1, 6). (The immune boosting effects has been thought to be associated to Vitamin C’s enhancement of non-heme iron (6); although there is research opposing this view (7).) Vitamin C deficiency, as is well known, causes scurvy which is typically seen after 1 month of dietary deficiency largely due to impaired collagen biosynthesis (1). Unfortunately, despite the importance of Vitamin C, studies attempting to show that supplementation was advantageous have been equivocal (2). For one, vitamin C absorption from oral administration appears to be highly regulated such that amounts ingested over normal intake appear to be excreted (1). (Of recent note, interest in intravenous vit C has shown some potential benefits) (2). More over, there are complex interaction Vitamin C has with trace metals that may confound outcomes (see below, and in 7).

Vitamin C is synthesized endogenously in most animals, however humans and primates have lost this capability, and so for us it is an essential dietary component (1). The USDA Dietary Reference Intakes for Vitamin C indicate that the Recommended Daily Allowance for males 19+ years old is 90 mg and females of the same age range is 75 ug/d. Of note, for smokers, there is an additional 35 mg/day recommended. Food sources of vitamin C include fruits and vegetables, especially citrus, tomatoes, potatoes, red and green peppers, kiwifruit, broccoli, strawberries, Brussels sprouts, and cantaloupe. According to the 2001–2002 National Health and Nutrition Examination Survey (NHANES), mean intakes of vitamin C are 105.2 mg/day for adult males and 83.6 mg/day for adult females, meeting the currently established RDA for most nonsmoking adults.

However, large variability, excluding significant environmental factors, have been found in circulating ascorbic acid in populations, and significant differences in response to dietary vitamin C noted (3). As mentioned in the assigned paper (4), single chain polymorphisms (SNPs) in the detoxifying enzyme glutathione S-transferase M1 (GSTM1) may impact ascorbic acid function and have been associated with serum ascorbic acid concentrations (3). GSTs containing glutathione act synergistically with ascorbic acid as antioxidants and these agents seem to protect each other from oxidation (3), thus being a potential rationale for maintaining serum ascorbic acid levels. Although studies have been inconsistent, one study suggested that the presence of the SNP in those who did not meet the RDA had a 4-12-fold greater risk of ascorbic acid deficiency than those without the SNP (3). This SNP appears wide spread with evidence that this mutation may be in about 50% of the population (4).

Authors Garry Buettner and Beth Anne Jurkiewicz highlight the potential adverse impact of low ascorbic acid serum concentration in these people with this SNP. There is a “cross-over” effect for ascorbate in the presence of catalytic metals from pro-oxidant at low concentrations to anti-oxidant at higher concentrations (7 with my italics). This suggests that if you recommend vitamin C supplements, that you avoid supplements with trace irons or other metals like silica. Additionally, looking for reasons for low levels of serum vitamin C may be justifiable given the pro-oxidative effects that may result.

Although vitamin C deficiency is not typically seen as a common public health problem, there are some reports that vitamin C deficiency remains an issue (5) The identification of the SNP described above may need more research, however appears to be an interesting line of clinical investigation.

(1)        NIH Vitamin A Fact Sheet for Health Professionals: https://www.nal.usda.gov/fnic/ascorbic-acid-vitamin-c

(2)        Yan MA et al. High-Dose Parenteral Ascorbate Enhanced Chemosensitivity of Ovarian Cancer and Reduced Toxicity of Chemotherapy. Science Translational Medicine  05 Feb 2014: Vol. 6, Issue 222, pp. 22

(3)        Da Costa, et al. Genetic Determinants of Dietary Antioxidant Status. Progress in Molecular Biology and Translational Science Volume 108, 2012, Pages 179-200

(4)        Costa V et al, Nutritional genomics era: opportunities toward a genome-tailored nutritional regimen. Journal of Nutritional Biochemistry 21 (2010) 457–467

(5)        Scurvy is a serious public health problem. Slate.com, Nov 20, 2015: http://www.slate.com/articles/health_and_science/medical_examiner/2015/11/scurvy_is_common_and_should_be_diagnosed_and_treated.html

(6)        Vitamin C and iron interactions:

a.         Haliberg L etal. The role of vitamin C in iron absorption. Int J Vitam Nutr Res Suppl. 1989;30:103-8.

b.         Morris ER. An overview of current information on bioavailability of dietary iron to humans. Fed Proc. 1983 Apr;42(6):1716-20

c.         Lynch SR et al. Interaction of vitamin C and iron. Ann N Y Acad Sci. 1980;355:32-44.

(7)        Garry R. Buettner and Beth Anne Jurkiewicz. Catalytic Metals, Ascorbate and Free Radicals: Combinations to Avoid. RADIATION RESEARCH 145, 532-541 (1996)

(8)        Cook JD. Effect of ascorbic acid intake on nonheme-iron absorption from a complete diet. Am J Clin Nutr. 2001;73:93–8.

Assessing The Digestive Systems

Assessing The Digestive Systems

The digestive system is critically important to human health and well-being.  It is also the mechanism of obtaining all nutrients.  As such, it is imperative to assess its functioning in every nutrition work-up. Any abnormality in the digestive system has the potential for causing mild to severe malnutrition.  According to  Allen Spiegel, Director of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), "Digestive diseases rank second among all causes of disability due to illness in the United States."   The range of digestive problems that may be encountered is varied and includes gastroesophageal reflux disease (GERD), irritable bowel syndrome, celiac disease, inflammatory bowel diseases, gastrointestinal cancer, ulcers, and gallstones.

Critical to an optimally functioning GI tract is a healthy mucosal barrier capable of regulating the particles crossing it. This, in turn, is dependent on the qualtiy of food consumed and the microflora living in the gut. Imbalance in these areas can result in varying degrees of intestinal hyperpermeabilty or leaky gut.  Once large molecules begin "leaking" from the gut into the bloodstream, it can result in an immune response to these antigens.  A reading in your assignments this week, explains this mechanism in greater detail (Gut. Oct 2006; 55(10): 1512–1520 (Links to an external site.)Links to an external site.).

Proper assessment, diagnosis, and therapy for digestive conditions can considerably improve quality of life, as well as nutrient status and health.  Later in the course, we will investigate assessments of various nutrients in the blood and other body fluids.  For this discussion, we will be looking primarily at how normal digestion and absorption can be impeded in an unhealthy GI tract and how we can best assess these functions.

Analysis of Urine

Analysis of Urine

In the normal adult, about 1200 ml of blood passes through the kidney each minute, exposing the plasma to the semi-permeable membrane of each functioning glomerulus. The ultra-filtrate that collects in Bowman’s capsule contains all of the substances of the plasma capable of passing through the membrane. Modification of this filtrate to produce excreted urine occurs in the tubules and collecting duct of the nephron. Threshold substances such as glucose and amino acids are reabsorbed. Waste materials such as creatinine, urea, uric acid, phosphates, and other materials are left in the filtrate to be excreted. The three principle factors affecting the composition of the excreted urine are nutritional status, the state of metabolic processes, and the ability of the kidney to selectively process the material presented to it.

Two of the most common assessments of kidney function are serum measurements of urea nitrogen (BUN) and creatinine.  Both of these substances are excreted entirely by the kidneys, so correlate well to kidney function.  However, it is important to consider that there are additional conditions that may also affect serum concentrations of these substances so, as always, results should be interpreted with consideration of the entire clinical picture.

Because urinalysis can screen for a variety of conditions and is a relatively easy specimen to obtain, it is a frequently performed assessment. Examination of both solute and urine sediment is done in the standard urinalysis. Among the most important conditions readily detected by chemical testing of urine solute are glycosuria, ketonuria, proteinuria, and the presence of the pigments bilirubin, urobilinogen, and hemoglobin. Proteinuria is probably the most common indication of renal disease. Microscopic examination of the urine sediment provides important information concerning the kidneys and urinary tract not readily obtainable in any other way. Urinary sediment can include red blood cells, white blood cells, epithelial cells, fat of biological origin, casts, bacteria, yeast, fungi, parasites, spermatozoa, crystals, and amorphous material. Some of these substances have pathological significance and some are not as clinically important.

In addition to the standard urinalysis, many other compounds can be measured in urine. For example, there is increased excretion of the organic acid formiminoglutamate (FIGLU) when folic acid is deficient. Similarly, xanthurenic acid increases when there is a lack of B6, which is necessary for its metabolism. There are many additional organic acids that can provide information with regard to nutritional deficiencies, dysbiosis, and inadequate detoxification. All of these metabolites are measured in urine. An overview of these organic acids is presented in this unit.  We will discuss many of these compounds in more detail as we progress through the course.

To understand how these organic acids can be helpful in identifying need for nutrient support, the diagram below may be helpful.  Note that the conversion of B to C is inhibited due to lack of a coenzyme. Therefore, if we know the coenzyme necessary for the reaction to occur, we can assume that providing that nutrient will enable the pathway to proceed in normal fashion.  This functional approach is often more sensitive than other assessments because it can identify those who may have increased needs due to lifestyle or genetic factors.

Discuss specific gravity, appearance, color, and pH of urine and the information we can learn from these assessments.

Discuss specific gravity, appearance, color, and pH of urine and the information we can learn from these assessments.

Urine color ranges from yellow to amber depending on urine concentration and the presence of pigment urochrome. From ancient times, the color of urine has been an important diagnostic tool, with 20 colors in 100 BC (3). Foods, medications, metabolic products and infections change the color (1). This article has pictures and descriptions of different colored urine.  (3)

Urine outside of the body will develop ammonia smell as bacteria break up urea molecules. Fresh urine with same smell often indicates infection with bacterial working in the bladder (2).

Urine specific gravity provides insight into patient’s hydration status and correlates with urine osmolality and the kidney’s capability in concentrating fluids (1). Normal urine USG is greater than 1.000 as the kidney removes water AND minerals, salts, and other compounds (1). The more additives to water, the higher the USG which normally can rise to 1.030 (2). Above 1.030 there is typically glucose or protein added, the syndrome of inappropriate antidiuretic hormone, or the patient’s is dehydrated (1,2). Overhydrated patients typically have USG value closed to water; other causes include diuretic use, diabetes insipidus, adrenal insufficiency, aldosteronism, impaired renal function (2).

Urine pH ranges from 4.5 to 8, and typically mirrors the plasma pH (except for cases of Renal Tubular Acidosis) (2). Typically, the serum (reflected in the glomerular filtrate) pH is around 7.4, and the kidneys with acidify to approximately 6.0 (1). The kidneys sensing and excreting excess alkali (bicarbonate or excess acid (hydrogen or ammonia) in the urine is a major way the body maintain acid-base balance (1). Other factors may cause variations in pH of urine: In cases of urinary tract infections, an elevated pH suggests the presence of bacteria that area splitting urea (2). More alkaline urine may be found in vegetarians who consume citrus fruits, legumes, and vegetables (1). Meat eaters, or those consuming cranberry juice, tend to have more acidic urine (1).

Urine pH is helpful in treating urine calculi: “Calcium phosphate, calcium carbonate, and magnesium phosphate stones develop in alkaline urine; when this occurs, the urine is kept acidic. Uric acid, cystine, and calcium oxalate stones precipitate in acidic urine; in this situation, the urine should be kept alkaline or less acidic than normal” (1).

(1)   RhCeus.com: Urinary ph. http://www.rnceus.com/course_frame.asp?exam_id=23&directory=ua

(2)   JEFF A. SIMERVILLE, M.D., WILLIAM C. MAXTED, M.D., and JOHN J. PAHIRA, M.D. Urinalysis: A Comprehensive Review. Am Fam Physician. 2005 Mar 15;71(6):1153-1162.

Profiling Amino Acids to evaluate vascular function.

Profiling Amino Acids to evaluate vascular function.

As far as Amino Acid profiling, there are limited ways to evaluate vascular function despite this system major impact in the leading causes of death and disability, namely heart attack and stroke. Arginine is involved in many human biochemical functions including ammonia detoxification, hormone secretion, and immune modulation. In vascular function, Arginine is a key player in Nitric Oxide (NO) function, which is a soluble gas modulating arterial system. Arginine’s effect via NO has implications for angina pectoris, congestive heart failure, hypertension, coronary artery disease, preeclampsia, intermittent claudication, and erectile dysfunction. (5).

Arginine can be derived from diet, however mostly the body produces the 80-120 umol/L concentrations endogenously from the amino acid citrulline. Citrulline is involved in the Krebs Cycle and is a by product of glutamine metabolism. Citrulline is taken up in kidney where it is converted in Arginine.(5) Arginine and oxygen combine to form Nitric Oxide and Citrulline, and the rate of production governed by Nitrous Oxide Synthetase found in endothelium, neural tissue, macrophages and neutrophils (3). Of note, the specific inducers of NOS in the immune cells includes endotoxins (or lipopolysaccharides) (3, 4). The action of NO is mediated by the free radical oxidant properties of the gas, and also by its activation of guanylate cyclase leading to relaxation of smooth muscle in blood vessel walls (3,4). Pharmaceutically, we see the results of NO action with nitroglycerin effect during myocardial infarct (3).

Arginine is also the precursor for the synthesis of proteins, urea, creatine, and agmatine (5). As the precursor of NO, arginine is involved in many physiological effects of the cardiovascular system, and is critical for maintenance of blood pressure, myocardial function, inflammatory response, and oxidative processes (5). Due to the various processes it is involved in, Arginime has been called one of the most versatile amino acids (2). There are clinical applications described that include HIV/AIDS, cardiovascular conditions, preeclampsia, growth hormone and athletic performance, burns and critical care, cancer, GI and GU conditions, perioperative nutrition, and preterm labor.(5). Safety studies have shown that this amino acid is safe in typically recommended doses of 1-15 g per day.

Sources: 

(1)  Clinical Guidelines for Determination of Preferred Specimen Choice by Bradley Bongiovanni, ND and Judy Feinerman, MS, RD Townsend Letter for Doctors and Patients: the Examiner of Medical Alternatives December 2003 Issue # 245, 38-42.)

(2)   Morris, S.M., 2007. Arginine Metabolism: Boundaries of Our Knowledge. The Journal of Nutrition 137, 1602S-1609S. https://doi.org/10.1093/jn/137.6.1602S

(3)  Biochemistry

(4)   McCann, S.M., Licinio, J., Wong, M.L., Yu, W.H., Karanth, S., Rettorri, V., 1998. The nitric oxide hypothesis of aging. Exp. Gerontol. 33, 813–826.

(5)  Arginine: Clinical Potential of a Semi-Essential Amino Acid. Alternative Medicine Review Volume 7, Number 6 ◆ 2002

Clinical Assessment and Protein Status

Clinical Assessment and Protein Status

Clinical assessment consists of a medical history and a physical examination to detect physical signs (observations made by the examiner) and symptoms (manifestations reported by the patient) associated with poor nutritional status.  It is very common for our clients to present with multiple health challenges that result from metabolic imbalances related to sub-optimal diet and lifestyle choices.  As we collect information, we want to identify a chief complaint to help direct us in further investigation and, ultimately, appropriate recommendations.  Very often, by supporting a client in resolving their chief complaint, many related issues will resolve as well.

It is important to be aware of subtle signs of sub-optimal nutrition. As you first meet your client, be alert to visual clues such as loss of muscle mass (floppy” triceps, “saggy” gluteal muscles, and overall muscle definition), skin tone (clear with healthy coloring, wrinkled, elasticity, rashes, blemishes), hair (quality, thickness, texture), and nails (shape, color, texture).  Two slide shows are presented in your assignments with photos of some signs of sub-optimal nutrition. 

Although many of the severe symptoms associated with nutrient deficiencies are not commonly observed in the United States, it is important for nutritionists to be aware of them.  It should also be kept in mind, however, that many of the critical physical signs are nonspecific and must, therefore, be interpreted in conjunction with biochemical, anthropometric, and dietary data before specific nutritional deficiencies can be identified. It is also helpful to be aware of the population groups at risk for some conditions. For example, although most Americans have adequate protein intake, it is still very possible to observe the symptoms of protein-energy malnutrition in certain population groups, such as those with AIDS, cancer, and eating disorders such as anorexia. Assessing protein and micronutrient status has also become a factor in follow-up of those patients who have undergone gastric bypass surgery.

Proteins are essential for structural and regulatory functions, as well as acting as specific protein carriers and mediators of the immune response. There are no dispensable protein stores in humans, and therefore loss of body protein results in loss of essential structural elements as well as impaired function. Most of the body protein is found in the skeletal muscle (somatic protein) and in the smaller visceral protein pool which includes serum proteins, blood cells, and solid tissue organs such as liver, kidneys, pancreas, and heart. These are the metabolically active protein. The other noncellular protein components of the body found in the connective tissue are not readily exchangeable with other body pools of protein. Therefore, during protein malnutrition and disease, alterations occur in somatic and visceral proteins, but not in the extracellular connective tissue.

Recently, there has been greater emphasis on testing individual amino acids or groups of amino acids, especially for those practicing functional medicine.  Some of this testing has been done routinely for years.  For example, blood spot testing of phenylalanine is a routine screening test for phenylketonuria (PKU) performed on newborns.  Newer applications for amino acid assessment include attempting to identify individuals in need of supplementation of essential amino acids or other therapies to correct abnormal amino acid status. 

Review of RMR.

For me it was an opportunity to investigate what constitutes energy in the body. As you point out calorimetry is basically O2 in and CO2 out giving an estimate of the amount of energy utilized. This to me points out that the estimate is based on mitochondrial function where O2 is taken in (with glucose) and CO2 produced (with ATP). However I did find an interesting article, “Beyond mitochondria, what would be the energy source of the cell?” which challenges the assumption that this is the only energy produced (1). In our basic texts, the workings of the mitochondria seem complex but understood. 

This article points out that “For the apparently well studied metabolic process Krebs cycle, which was described as early as 1937 and is found in nearly every biology and chemistry curriculum, there is a considerable disagreement between at least five databases. Of the nearly 7000 reactions contained jointly by these five databases, only 199 are described in the same way in all the five databases. Thus to try to integrate chemical energy from melanin with the supposedly well-known bioenergetic pathways is easier said than done; and the lack of consensus about metabolic network constitutes an insurmountable barrier.”

These authors concluded based on research of research on melanins ability to transform photons of light into chemical energy that melanin may represent over 90% of cell energy requirements (!).

Herrera, Arturo S., Maria Del C A Esparza, Ghulam Md Ashraf, Andrey A. Zamyatnin, and Gjumrakch Aliev. “Beyond Mitochondria, What Would Be the Energy Source of the Cell?” Central Nervous System Agents in Medicinal Chemistry 15, no. 1 (2015): 32–41.

Adipose tissue association with inflammation, and the development of insulin resistance

The authors in the review paper assigned this week, “Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes” (1) summarize data that implicate an immune mechanism as a possible cause of insulin resistance and type 2 diabetes in obese individuals. A concluding remark suggests that should further research confirm that diabetes is an inflammatory disease that anti-inflammatory therapies could have a role in prevention and treatment. Of note, immune modulating therapies and monoclonal antibodies are a major focus of new research (2).

Interestingly, for this course in nutritional measurement, the paper notes that WBC, fibrinogen, CRP are all elevated obese diabetic patients. All of these are easily and inexpensive to measure and may be useful from a nutritional viewpoint in identifying patients at risk for progressive chronic conditions.

Insulin resistance seems to be linked to inflammation by the finding that TNF alpha is increased in tissues targeted by insulin hormone (adipose, liver, muscles). Other immune factors and monocyte infiltration are also noted in these tissues in obese individuals. This review provides a window into an association between obesity, insulin resistance/diabetes, and inflammation.

Obviously, the obesity-diabetes-inflammation link is a complex one, and the assigned article, “Fat – An Evolving Issue” shows this complexity just in obesity alone (3). “Three controversial suggestions” are described as factors in obesity – “gut microbiome, stress and endocrine-disrupting chemicals”. Although these factors seem to be downplayed, I suspect these are more of an influence in obesity than they are given credit for in this article. I found it very encouraging to find an article, by a geneticist(!) in 2011 which in its first sentence said to my amazement that “One of the most unexpected and fertile advances in biology engendered through mouse genetics has been the rediscovery that physiology has to be studied ultimately at the level of the entire organism.” (4, my emphasis) The research described shows how Serotonin in the gut plays a major role in inhibiting bone formation. Interestingly one of the other assigned articles on sarcopenia deals with this topic of “bone loss” (5).

I am very interested in the implications of Hans Selye’s stress paradigm and new findings in stress research. For instance, Serotonin has been found to have widespread impact on an organism, whether on the gastrointestinal tract, the platelets, the immune system, or the brain. Although there a many interesting articles on stress, as related to diabetes, serotonin, and inflammation, I found this article particularly relevant, “Serotonin as a New Therapeutic Target for Diabetes Mellitus and Obesity” (6): “In peripheral tissues, suppressing 5-HT signaling might represent a new target for anti-obesity treatment by increasing energy expenditure and improving insulin resistance”.  In nutritional interventions, perhaps there is a link between stress and foods that “irritate” the GI tract (causing endotoxemia (7)) prompting the release of serotonin leading to insulin resistance and diabetes.

(1)    Esser, Nathalie, Sylvie Legrand-Poels, Jacques Piette, André J. Scheen, and Nicolas Paquot. “Inflammation as a Link between Obesity, Metabolic Syndrome and Type 2 Diabetes.” Diabetes Research and Clinical Practice 105, no. 2 (August 2014): 141–50. https://doi.org/10.1016/j.diabres.2014.04.006.

(2)    https://labiotech.eu/top-5-immunotherapies-synteracthcr/

(3)    Speakman, John R., and Stephen O’Rahilly. “Fat: An Evolving Issue.” Disease Models & Mechanisms 5, no. 5 (September 2012): 569–73. https://doi.org/10.1242/dmm.010553.

(4)    Karsenty, Gerard, and Michael D. Gershon. “The Importance of the Gastrointestinal Tract in the Control of Bone Mass Accrual.” Gastroenterology 141, no. 2 (August 2011): 439–42. https://doi.org/10.1053/j.gastro.2011.06.011.

(5)    Budui, Simona L., Andrea P. Rossi, and Mauro Zamboni. “The Pathogenetic Bases of Sarcopenia.” Clinical Cases in Mineral and Bone Metabolism: The Official Journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases 12, no. 1 (April 2015): 22–26. https://doi.org/10.11138/ccmbm/2015.12.1.022.

(6)    Oh, Chang-Myung, Sangkyu Park, and Hail Kim. “Serotonin as a New Therapeutic Target for Diabetes Mellitus and Obesity.” Diabetes & Metabolism Journal 40, no. 2 (2016): 89. https://doi.org/10.4093/dmj.2016.40.2.89.

(7)    Pomytkin, Igor A., Brandon H. Cline, Daniel C. Anthony, Harry W. Steinbusch, Klaus-Peter Lesch, and Tatyana Strekalova. “Endotoxaemia Resulting from Decreased Serotonin Tranporter (5-HTT) Function: A Reciprocal Risk Factor for Depression and Insulin Resistance?” Behavioural Brain Research 276 (January 2015): 111–17. https://doi.org/10.1016/j.bbr.2014.04.049.

Another important anti-oxidant, Uric Acid

Another important anti-oxidant, Uric Acid, to the mix and show how vitamin E helps maintain anti-oxidant rather than "gouty levels" of uric acid in blood. 

Uric acid via the p53 gene (tumor suppressor) and GLUT-9 uric acid transporter is theorized to be another powerful intracellular anti-oxidant (1). In fact, high uric acid levels in higher primates/humans may be reason for longer life span (1). Of course, uric acid also related to the development of gout (1). 

By supplementing with vitamin E, one study I found showed that in rats, there was an increase in urinary excretion of uric acid (2). Possibly, the vitamin E allows the cells to maintain level of anti-oxidation which allows reduction in serum uric acid levels? Supplementing with vitamin E may be a consideration then for patients on gout treatment.

1/Itahana Y et al. The uric acid transporter SLC2A9 is a direct target gene of the tumor suppressor p53 contributing to antioxidant defense .Oncogene volume 34, pages 1799–1810 (02 April 2015).

2/Seifi B1, Kadkhodaee M, Zahmatkesh M.. Effect of vitamin E therapy on serum uric acid in DOCA-salt-treated rats.. Acta Physiol Hung. 2011 Jun;98(2):214-20. doi: 10.1556/APhysiol.98.2011.2.13.

Nitric Oxide Theory of Aging

NO does appear to have beneficial qualities in allowing blood vessel dilation and increase blood flow to occur at needed times. On the other hand, these effects seem to be needed at times in cases due to disruption in normal function - such as by viral or endotoxin exposure (1,2). The NO theory of aging  suggests that repeated infections create progressive oxidative damage leading to coronary disease and neurodegenerative conditions (1,2). The principal mechanism tested is when bacterial lipopolysaccharides (LPS) injected, NO Synthetase is induced (via an IL-1) mediator within 2-4 hours (2). The blood brain barrier does not appear to keep LPS out of the central nervous system (3), and an intense NO response is seen in the brain (1,2). The effect of NO is to inactivate enzymes which leads to cell death of invading virus as well as host cells (1). Antioxidant vitamin C and E appear to be beneficial in attenuating the NO oxidant effect (1,2). 

Sources:

1/ McCann S et al. The nitric oxide theory of aging revisited. Ann N Y Acad Sci. 2005 Dec;1057:64-84.

2/McCann S et al. The nitric oxide hypothesis of aging. Exp Gerontol. 1998 Nov-Dec;33(7-8):813-26.

3/Wei-Ye L et al. Increasing the Permeability of the Blood–brain Barrier in Three Different Models in vivo. CNS Neuroscience & Therapeutics. 21 (2015) 568-574.

a) Where and how are superoxide radicals generated? b) Do they have constructive, destructive or both functions?

a) Where and how are superoxide radicals generated? b) Do they have constructive, destructive or both functions?

A free radical is “an atom, molecule, or molecular fragment that has one or more unpaired electrons” which leads to its “high reactivity” and “pro-oxidant activity” (1). If these radicals are oxygen-based, they are reactive oxygen species (ROS); if nitrogen based then reactive nitrogen species (RNS) (1). Oxygen is the terminal electron acceptor in mitochondria for energy production. However, high levels of intracellular oxygen can be harmful to cell as oxygen molecules can become incomplely reduced, termed reactive oxygen species. These ROS include superoxide radical ion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH-) (2).

ROS reactions take place in multiple locations within a cell: the plasma membrane, nucleus, vacuole, or mitochondria (1). When these processes lead to excessive ROS then this is termed oxidative stress (1). Processes that contribute to ROS include (1):

1. Environmental pollution, including chemical, drugs, radiation, pollution.
2. Exposure to very high levels of oxygen
3. Normal physiological responses
   1. Oxidative phosphorylation in mitochondria
   2. Immune defense
   3. with liver metabolism (peroxisomes and cytochrome P450).

Superoxide radicals may be formed when molecular O2 has a reaction, and may be seen with CONSTRUCTIVE reactions such as with (1):

• tetrahydrofolate
• with the electron transport chain
• Cytochrome P450 enzymes
• white blood cells

With excessive reactions or toxins present, superoxide radicals can react to form secondary reactive substances like  hydrogen peroxide (H2O2) and hydroxyl radical (OH-) (1). Specifically, the hydroxyl radical can be harmful as it will react quickly and definitely with surrounding molecules, including DNA molecules (1). These harmful reactions can lead to DNA damage or protein degradation that can impair cellular function and lead to disease state.s 

Sources:

1/ Gropper et al. Advanced Nutrition in Human Metabolism. 7th Ed. Cengage. 2018.

2/ Thorpe, G.W., Reodica, M., Davies, M.J., Heeren, G., Jarolim, S., Pillay, B., Breitenbach, M., Higgins, V.J., Dawes, I.W., 2013. Superoxide radicals have a protective role during H 2 O 2 stress. Molecular Biology of the Cell 24, 2876–2884. https://doi.org/10.1091/mbc.e13-01-0052 (Links to an external site.)Links to an external site.

Points about the sensation of fatigue or exhaustion

Tim Noakes, a researcher and ultra-marathon runner, has authored many papers on a theory called, the Central Governor Model of fatigue. In this theory, fatigue is similar to thirst or hunger in that there is a monitor in the central nervous system that regulates the sensation (2). For example, hypoglycemia during exercise may elicit a fatigue feeling from the nervous system. By exposing the nervous system to hypoglycemia repeatedly, as in training for a marathon, the nervous system may lower its threshold for the fatigue alarm of hypoglycemia. (Perhaps this is also the explanation for the "Keto Flu (Links to an external site.)Links to an external site." that occurs for first few days of ketogenic diet as the body gets used to a new set point for serum glucose.) This theory is in opposition to the Energy Depletion Model that was first proposed by cardiologist AV Hill (1):

"In 1923, Nobel Laureate Archibald V Hill developed the currently popular model of exercise fatigue. According to his understanding, fatigue develops in the exercising skeletal muscles when the heart is no longer able to produce a cardiac output which is sufficient to cover the exercising muscles' increased demands for oxygen. This causes skeletal muscle anaerobiosis (lack of oxygen) leading to lactic acidosis. The lactic acid so produced then ‘poisons’ the muscles, impairing their function and causing all the symptoms we recognise as ‘fatigue’."

Summarizing Noakes theory, there are 2 types of fatigue - one is peripheral, so that when you are lifting weights at some point you are fatigued and muscle can't go further. Then there is second type which is central and relates to the exhaustion or hitting the wall when doing endurance exercise. In the article that you can link to below it talks about "pacing" as a factor in the experience of fatigue (1):

"...complex brain mechanisms are able to determine the physical state of the body on a moment to-moment basis and to adjust the work output specifically to insure that exercise can be conducted safely without the development of a catastrophic biological failure."

(Figures from source 2.)

My interpretation is that it seems that the purpose of the exercise and the confidence in the exerciser is an important element leading to success or exhaustion. There is interesting evidence for both the Energetic Depletion Model and the Central Governor Theory of fatigue.

Source (1): 

Noakes, T.D., 2012. The Central Governor Model in 2012: eight new papers deepen our understanding of the regulation of human exercise performance. British Journal of Sports Medicine 46, 1–3. https://doi.org/10.1136/bjsports-2011-090811 (Links to an external site.)Links to an external site.

https://bjsm.bmj.com/content/bjsports/46/1/1.full.pdf

(2) Noakes, T.D., 2012. Fatigue is a Brain-Derived Emotion that Regulates the Exercise Behavior to Ensure the Protection of Whole Body Homeostasis. Front Physiol 3, 82. https://doi.org/10.3389/fphys.2012.00082 (Links to an external site.)Links to an external site.

https://www-ncbi-nlm-nih-gov.revproxy.brown.edu/pmc/articles/PMC3323922/?tool=pubmed