LAB 9: Vitamin A Assessment 2014

LAB 9: Vitamin A Assessment 2014 Facts and Definitions Vitamin A - generic term for all related naturally occurring compounds that qualitatively exhibit some degree of biological activity of all-trans Retinol Retinoids any compound (naturally occurring or synthetic analog), that exhibits vitamin A activity. Food sources: pre-formed vitamin A - found only in foods of animal origin (e.g. retinol and retinyl esters). pro-vitamin A forms - can be metabolically converted into retinol by the body. Found in some plants and to a limited extent in some animal products (e.g. β-carotene and a few other carotenoids). other sources: synthetic analog forms retinoid-based drugs e.g. Accutane, (used in the treatment of cystic acne); Etretinate and Tigason, (used in the treatment of psoriasis and other dermatologic conditions). Principle forms of vitamin A found in the body: (1) Retinol (the alcohol form) most abundant form in the circulation. (2) Retinyl esters (in multiple forms) serve as the main storage form of the vitamin (3) Retinoic acid (the active form) serve as a hormone involved in gene regulation Tissue distribution of principle forms: Liver - (normally > 85% of body total). Majority as retinyl esters. Extrahepatic tissues (particularly fat typically 8-12% of body total). Almost all as retinyl esters. Plasma/Serum - ~1% of the body s vitamin A total. Retinol as major form with other minor forms Dietary Vitamin A and Retinol Equivalents Amount / Form Consumed Retinol Activity Equivalence (RAE) 1 µg Retinol (Dietary or in a Supplement) = 1 µg RAE 1 µg Retinyl Ester (Dietary or in a Supplement) = 1 µg RAE 2 µg β-carotene (in a Supplement) = 1 µg RAE 12 µg β-carotene (Dietary) = 1 µg RAE 24 µg α-carotene (Dietary) = 1 µg RAE 24 µg β-cryptoxanthin (Dietary) = 1 µg RAE u NOTE - Of the over 600 carotenoids and related xanthophylls found in nature, < 60 have any vitamin A activity at all. [Although some can be absorbed and enzymatically converted to retinol, the process is so inefficient that they would have an equivalence ratio > 100 µg (some > 500 µg) = 1 µg RAE a quantity that may be impossible to consume in the context of a diet]. u There is also relatively sparse data with respect to changes (particularly losses) that occur as a result of ripening, storage and cooking of vitamin A-containing food sources. u Synthetic, therapeutic retinoids (e.g. Accutane, Tigason, Etretin and Etretinate) possess varying degrees of specific vitamin A activity (related to gene regulation) thus making them highly effective for dermatologic and other conditions. But, these therapeutic forms typically do not have any activity in vision or some of the other classic vitamin A functions. And, while neither a dietary component nor a supplement, these retinoids will affect how endogenous vitamin A forms are stored, metabolized and transported. Main targets for vitamin A assessment : (1) EYE as a target tissue (examining both functional and histological signs) (2) PLASMA / SERUM as surrogate biomarker for stores and homeostatic regulation (conc. & metabolites) (3) DIET - sources, quantities and bioavailability

LAB 9: Vitamin A Assessment 2014 Progression of Vitamin A Deficiency and Status Indicators u A progressive series of eye-related effects can be observed as an individual progresses from normal to marginal to depleted and ultimately deficient status. These effects have been correlated to levels in the body. Vit. A STORES PLASMA LEVELS BIOCHEMICAL EFFECTS HISTOLOGICAL EFFECTS Adequate Adequate None = Normal Vision None = Normal Histology ( > 0.1 µm) ( > 1.05 µm) [ 1.05 0.1 µmol/g Liver ] [3.0-1.05 µm] ( no evidence of functional deficit ) [~900-300 ng/ml] Marginal g Depleted Marginal g Depleted Impaired Dark Adaptation None = Normal Histology (0.1 g 0.07 µmol /g Liver) (0.7 g 0.35 µm) g g Night Blindness g g abnormal* [~200-100 ng/ml] *(up to 10% of individuals may even exhibit Bitot s spots) Depleted g Deficient Depleted g Deficient worsening vision Bitot s Spots g Xerosis g (< 0.07 µmol /g Liver) ( < 0.35 µm) g Keratomalacia g Blindness [<100 ng/ml] (>85% of individuals display Xerophthalmia) u Xerophthalmia - clinical term for the dry eye and accompanying histopathologic ocular effects on the eye, which range from Bitot's spots (an early / mild sign) to conjunctival and corneal xerosis (intermediate to later sign) and eventually keratomalacia (late/severe). u NOTE - The length of time it takes to develop these signs is a function of body reserves, intake and an individual s body needs. With adequate reserves, an individual may remain symptom free for many months in spite of meager intake. If, on the other hand, reserves are very low, the onset and progression to severe symptoms can occur very rapidly (within a few weeks) once dietary intake becomes low or ceases. u Excess and Toxicity can also be an issue Liver Retinol conc. plasma/serum Retinol conc. Associated / correlated with > 1.1 µmol/g Liver > 3.0 µmol/l (µm) Excess / Toxicity Vitamin A Homeostasis and RDR tests An issue when evaluating plasma/serum vitamin A is that with adequate stores, the circulating level is homeostatically controlled and is thus relatively constant over a wide range of intakes. This occurs because the liver adjusts to changes in intake by either putting more into storage (esterifying Retinol by adding a fatty acid residue g g retinyl esters) when intake exceeds body needs, or mobilizing retinol from storage (removing the fatty acid group, thus regenerating retinol) into the circulation for transport to tissues during times of need. Because of the liver s dominant role as a vitamin A storage organ, determining liver levels would be the ideal way to assess vitamin A status if getting a liver sample was an option. The Relative Dose Response (RDR) test uses plasma/serum retinol in a manner that has proven to be reliable as a surrogate for assessing liver vitamin A stores and identify individuals with marginal to severe vitamin A deficiency. In the RDR test, an initial, baseline fasting blood sample is taken first, and then immediately followed by administration of a small oral dose of vitamin A. Next, a small, high fat snack is given to ensure and facilitate rapid vitamin A absorption. If an absorption problem is suspected, the vit. A dose can be administered I.V. After 5 hours, a second blood sample is obtained. The concentration of plasma/serum retinol is determined in the before (0 hr.) and after (5 hr) samples.

LAB 9: Vitamin A Assessment 2014 Relative Dose-Response (RDR) [%] = Plasma Retinol @ 5 hr. - Plasma Retinol @ 0 hr. x 100 Plasma Retinol @ 5 hr. Normal (vitamin A adequate) = 0-14 % Mild to Severe deficiency = 20 % Marginal status* = 15-19 % * NOTE - the range of 15-19% is a gray area that usually indicates Marginal status (and thus could be indicative of early or impending deficiency). However, there are instances when normal (adequate) individuals fall in this range. Thus, individuals identified as having marginal status should be retested. Modified Relative Dose Response Test The Modified Relative Dose Response (MRDR) is simpler and less burdensome to subjects because it requires only 1 blood sample. For the MRDR test, a small test dose is administered (as in the RDR test) except that the retinoid used is 3,4-didehydroretinyl acetate (ddract). This didehydro- compound is naturally occurring (originally obtained from certain fresh water fish), but not typically found in humans to an appreciable extent. Following absorption, the ddract is metabolized to 3,4-didehydroretinol (ddroh). Because ddroh is highly similar to the retinol (ROH) endogenously found in humans, it is transported and metabolized identically. Thus, after a 4-6 hr period following ddract administration, a single blood sample is taken. The sample is processed and analyzed as in the standard RDR test, except that both retinol (ROH) and ddroh are measured. The ratio of [ddroh]:[roh] is a measure of the the body s response. Vitamin A Status MRDR = Ratio (µm ddroh : µm ROH) Adequate <.03 Marginal status.03 -.06 Deficiency >.06 * NOTE - 3,4-didehydroretinyl acetate (ddract) is not typically found in humans except in situations where the diet includes high consumption of specific fresh water fish that contain high levels of didehydro- retinoids. Thus, the MRDR should not be used if the individual or population is known to have recently consumed didehydroretinyl-rich fish. If such consumption is not taken into account, the ratio could be significantly affected because of the already elevated levels of ddroh in the circulation (resulting in a ratio >.06) and misidentification of individuals as deficient. Factors affecting Vitamin A levels or homeostasis Factors or situations => DECREASE in Serum / Plasma Retinol (1) Infection (2) Chronic Inflammatory conditions (3) Iron deficiency (sometimes) (4) Zinc deficiency (5) Protein Energy Malnutrition (6) Therapeutic retinoids (7) Liver disease (including alcoholic cirrhosis) Factors or situations => INCREASE in Serum / Plasma Retinol (1) Renal disease (2) Estrogens (as treatments) u NOTE - these factors can impact RDR results, and thus should be taken into account in RDR interpretation. Vitamin A Measurement Utilizes HPLC (High Performance Liquid Chromatography), wherein the various vitamin A forms/metabolites can be separated and quantitated individually. Plasma retinol reflects vitamin A status only when body stores are severely depleted or are very high. Issues with this methodology include the chemical nature of the retinoids themselves. Vitamin A and its metabolites (particularly Retinol and Retinoic acid) are very sensitive to oxidation and destruction by light and heat. Failure to adequately protect samples from heat and light during collection, transport, storage and processing can lead to significant errors in their measurement.

Name: Lab TA: Week 9 - PROBLEM SET ( 125 points ) Note: Clinical values are reported to two decimal place unless otherwise specified; please report your final calculations accordingly. Must show calculations and units for full credit. Consult the supplied Appendix for problem 11. (1) With respect to vitamin A and its multiple forms and metabolites, indicate whether the following statements are either True or False by writing either T or F next to each statement. (5 points) Retinol and retinyl palmitate are examples of preformed vitamin A. All plant-derived carotenoids are provitamin A forms. Retinoids can only be obtained from foods. Accutane is a retinoid with some vitamin A activity. Accutane is a retinoid with some vitamin A activity. (2) The following are serum vitamin A values as part of the RDR test from 4 participants. Use the information provided to Calculate RDR for each participant and indicate if the participant s vitamin A stores are A = adequate, M = marginal, D = deficient. Show your work for full credit. (17 points) Participant Time 0 serum Vit. A conc. (ng/ml) 5 hour serum Vit. A conc. (ng/ml) A 288 321 B 417 533 C 376 455 D 198 331 Participant RDR calculation RDR (%) A Vitamin A: A = adequate M = marginal D = deficient B C D c) Which participant(s) would be most likely to exhibit night blindness and Bitot s spots? d) One participant does display Bitot s spots as well as early signs of conjunctival xerosis. Which participant would you most likely expect to display these signs? e) Provide justification for your answer to part (d).

Name: Lab TA: (3) A small village in southern Africa has been under observation for several years with respect to their vitamin A status. Average values for all members of the village are given below. (19 points) a) Calculate the January 2013 RDR average and standard deviation from the following participant tests and fill in the table below (determine the average of the 5 samples). Average values for all members of the village over time Space to show work for mean and standard deviation calculations. Date Average RDR (StdDev) January 2009 15.84% (± 2.7%) June 2009 11.87% (± 3.4%) January 2010 16.22% (± 2.6%) June 2010 12.71% (± 3.2%) January 2011 18.36% (± 2.7%) June 2011 16.42% (± 3.1%) January 2012 18.91% (± 2.8%) June 2012 17.26% (± 3.3%) January 2013 Participant time 0 vit.a (ng/ml) 1 322 401 2 342 413 3 314 399 4 323 408 5 327 392 Individual participant tests from January 2013 5 hour vit.a RDR calculation RDR (%) (ng/ml) b) Rainfall and crop records indicate that 2009 and 2010 were good years (above average rainfall and above average crop yield), while 2011 and 2012 were just average with respect to rainfall and crop yield. The most recent 2013 crop cycle had below average rainfall and initial crop yields are below average. Climate projections for the region suggest below average rainfall for at least the next crop cycle as well. Based on the vitamin A status data above, and the crop and weather projections, is vitamin A supplementation of the village warranted? (Yes or No) c) Briefly justify your answer.

Name: Lab TA: (4) An individual with a 4300 g. liver (wt.) has a liver vitamin A concentration of 0.52 µmol/g. Show your work and fill in your answers to the following question in the table below. (13 points) A) What is the estimated TOTAL quantity of Liver vitamin A stores? B) Using a liver vitamin A concentration of 0.07µmol/g as the threshold for deficiency, what is the quantity of Liver vitamin A stores at the threshold for deficiency? C) If this individual were to suddenly stop consuming any and all forms of vitamin A (a completely vitamin A deficient diet), estimate how many days would it take for them to reach the 0.07µmol/g Liver concentration threshold for vitamin A deficiency (and the onset of signs). Assume a normal, steady vitamin A utilization rate of 0.60% per day. Answers should be recorded as number of days to reach vitamin A deficiency threshold. D) Assuming all factors above are the same (Liver wt., utilization rate) BUT the starting Liver vitamin A concentration = 0.15 µmol/g Liver, estimate how long it would take to reach the 0.07 µmol/g Liver threshold concentration for deficiency. Answers should be recorded as number of days to reach vitamin A deficiency threshold. E) Assuming all factors above are the same (Liver wt., utilization rate) BUT the starting Liver vitamin A concentration = 0.092 µmol/g Liver. Estimate how long it would take to reach the 0.07 µmol/g threshold concentration for deficiency. Answers should be recorded as number of days to reach vitamin A deficiency threshold. Question Part A Show your work. Answer (include units) B C D E

Name: Lab TA: (5) Based on the typical symptomatic progression (from normal) to vitamin A deficiency, place the events or values below into the most logical chronological order (1 = first/earliest; 9 = last) (9 points) Squamous metaplasia detected from conjunctival impression cytology (evidenced by decreased number of goblet cells and increased number of abnormal, squamous epithelial cells) Plasma vitamin A level at 1.062 µm (~306 ng/ml) Evidence of night blindness together with an increased pupillary threshold score RDR test results give a value of 21.7% Evidence of conjunctival xerosis later sign. Impaired dark adaptation Corneal Keratomalacia present Plasma vitamin A levels drop from 1.02 µm to.53 µm (293 ng/ml to 152 ng/ml) in 3 months Bitot s spots appear (6) Five participants were administered the MRDR test 3 days after taking the standard RDR as part of a comparison study. Samples for the standard RDR test were collected at a remote field site, while the modified RDR (MRDR) test was performed on-site at a clinic lab. (23 points) a) Calculate each subjects MRDR from the values provided. Report answers to 2 significant digits. Participant ROH ddroh standard (µmol/l ) (µmol/l ) RDR (%) Calculation MRDR A 1.12 0.024 15.2 B 0.34 0.031 21.7 C 1.07 0.026 23.7 D 0.72 0.047 20.3 E 1.08 0.137 13.4 Complete the table below to answer the following questions: b) Based only on the MRDR, which participants (if any) have values indicative of Vitamin A deficiency? c) Based only on each participant s retinol (ROH) concentration above, which participant(s) would you have expected to be vitamin A deficient? d) Based only on the RDR test values above, which participants (if any) have values indicative of Vitamin A deficiency? e) For which participant(s) do all 3 indices (plasma ROH, MRDR and the RDR) indicate vitamin A adequate status? f) For which participant(s) do all 3 indices (plasma ROH, MRDR and the RDR) indicate vitamin A deficient status? Question Part Answer options (Instructions: circle all that apply for each question above.) (b) A B C D E None (c) A B C D E None (d) A B C D E None (e) A B C D E None (f) A B C D E None g) For the participants(s) with conflicting assessment indices, match the possible explanation below with the most likely subject for that explanation: Has been consuming a diet high in certain freshwater fish as their source of meat (no red meat or poultry) Their plasma sample was accidently exposed to heat (it was left in the trunk of the transport vehicle for 3 hours) prior to extraction and analysis.

Name: Lab TA: (7) Which of the following conditions or situations would alter normal vitamin A homeostasis or alter interpretation of vitamin A indice values? (2 points) a) Fat malabsorption or an extremely low fat diet b) Protein energy malnutrition c) Zinc deficiency d) All of the above e) a and b only (8) Which of the following would pose a significant risk of vitamin A toxicity to an otherwise well nourished, vitamin A sufficient individual? (2 points) a) Consuming a large serving of undercooked (very rare) or raw calf liver b) Taking Accutane over multiple days in succession c) Consuming multiple, large servings of sweet potatoes, butternut squash and carrots d) All of the above e) a and b only (9) A subject presents with signs of vitamin A deficiency including night blindness and Bitot s spots. Based on this, which of the following is/are most likely to be true? (2 points) a) The subject has a plasma retinol concentration of 1.16 µm (~334 ng/ml) b) The subject has an RDR value of 8.3% c) The subject has a plasma retinol conc. of 0.47 µm (~135 ng/ml) and an RDR value of 24% d) a and b only e) None of the above (10) A 20 yr. old female complains of frequent, persistent headaches, joint and muscle pain, and the feeling that their eyes are painfully swollen and bulging. A food log indicates a balanced diet with an adequate variety of fruits, vegetables and animal products such that her intake is ~800 RAE. The following are results of 2 RDR tests given 3 days apart. Calculate her RDR for both tests and state her vitamin A status based on their RDR values (A = Adequate; M = Marginal; D = Deficient). 0 hr. 5 hr. Test ROH ROH Calculation (ng/ml) (ng/ml) 1 1298 1307 2 1337 1341 RDR (%) (13 points) Vitamin A status: A / M / D b) In further discussions regarding her lifestyle habits, you learn that she thinks of herself as health conscious; worries about her skin and complexion (liberally using Retin-A containing skin products); had recently borrowed and took a 2 doses of her friend s Accutane. In addition, she takes a daily special skin rejuvinator liver oil-based supplement capsule (which contains 2000µg Retinyl Ester + 24,000 µg β-carotene). Given this new information and the data and information above, briefly discuss your conclusion about her vitamin A status, and what recommendations you would give her.

Name: Lab TA: (11) Mrs. Chapman (Mrs. C) is a 38 yr old female (5 5 / 160 lbs) who was recently diagnosed with Liver disease which is likely a consequence of her alcoholism. Because of her alcoholism, she does not eat very much throughout the course of the day. Currently, her vitamin A status is normal. Given below (see table) is an example of a typical daily dietary intake (food record) for Mrs. C. Her food intake was entered into a food database program (Food Processor) in which her % RDA for vitamin A was analyzed (see attached appendix). (20 points) MEAL FOOD ITEM QUANTITY MEASUREMENT Breakfast Black Coffee 3 cup Whiskey 1 cup Snack Baked Potato Chips 1 oz Lunch White Bread 2 slice Peanut Butter 2 Tbsp Strawberry Jelly 2 Tbsp Beer 12 oz (1 can) Snack Popcorn; butter flavored 1.5 cup Dinner Spanish Tomato Rice (homemade) 1 cup Refried Pinto beans 1 cup Flour Tortilla (Large) 1 each Colby cheese (shredded) 1 oz Cooked corn 1 cup Tomato Salsa 0.5 cup Red Wine 375 ml a) Complete the table below of Mrs. C s Vitamin A recommended and actual intake: Recommended intake for vitamin A (based on diet record Recommended Vitamin A intake (in RAE) Actual Vitamin A intake b) Complete the table below regarding Mrs. C s diet above. 1. 4. List sources of pro-vitamin A 2. 5. 3. List sources of pre-formed 1. vitamin A List foods that aid vitamin A 1. 2. absorption List foods that impair vitamin A absorption 1. 2. c) Answer the following question in the table below. Given Mrs.C s physiological condition and the assumption that she will maintain her dietary intake as indicated by her food record, will she become vitamin A deficient? (Yes or No) List the 2 most likely early ocular vitamin A deficiency signs Mrs. C 1. may be experiencing 2. If Mrs. C s alcoholism has significantly decreased her liver function (she has cirrhosis), how would this impact vitamin A metabolism and homeostasis, and thus your assessment of her vitamin A status?