Concentrations of Carotenoids,Retinol,and ... - Clinical Chemistry

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Nov 29, 1988 - Concentrations of Carotenoids,Retinol,and Tocopherolin Plasma, in Response to Ingestionof a Meal. Ellen D. Brown,AliceRose,NealCraft ...

CVs for the laser immunonephelometry-2% and 10%, respectively-compare well with those for techniques such as radioimmunoassay, enzyme-linked immunoassay, radial imniunodifflision, and inununoturbidimetry (5-7). The detection limit of 1 mg/L suffices for the screening of diabetic patients for microalbuminuria. Automatization of the method facilitated analysis of large runs of samples. Smaller runs, however, can also be handled easily and do not make the method more expensive. The low cost of the method is mainly due to the easy production of large amounts of antiserum, which need not be purified, and to the need for only small volumes of antiserum in the assay.Laser immunonephelometry costs about $30 per 100 samples, in duplicate, which can be performed in one 8-h working day. Analysis of the same number of samples measured by radioimmunoasaay would costabout $500. Urine is often stored for long periods at -20 #{176}C, particularly in research programs. We found albumin concentrations to be decreased in frozen urine samples, especially in those in which precipitates (mainly urates) had formed. Presumably, albumin is entrapped in the precipitates; however, heating five precipitate-containing samples for 1-12 h at 37 and 56#{176}C did not restore the albumin concentration to its original value. It has also been suggested that urinary pH is important in the formation of precipitate leading to decreased albumin concentrations in urine samples (8), but we found no relation between pH and precipitate formation. We found no loss of urinary albumin when samples were analyzed within two weeks and stored at 4#{176}C. Because important conclusions are sometimes drawn from rather small variations in albuminuria, one must be aware that storage at -20 #{176}C can influence albumin concentration. In a

concurrent study, Erman et al. (9) found a decrease in urinary albumin concentration after even as little as one week of storage at -20 #{176}C. We confirm that albumin concentrations are underestimated in frozen samples. Two of our samples stored at -20 #{176}C for two weeks showed substantial loss of albumin, but greater differences were seen after two and six months of frozen storage. References 1. AndersenAR, Christiansen JS, Andersen JK, et a!. Diabetic nephropathyin type 1 (insulin-dependent)diabetes: an epidemiological study. Diabetologia1983;25:496-501. 2. MogensenCE. Microalbuminuria as a predictor of clinical diabetic nephropathy. Kidney mt 1987;31:673-89. 3. Feldt-Rasmussen B, Mathiesen ER, Deckert T. Effect of two years of strict metabolic control on progression of incipient nephropathy in insulin-dependent diabetes. Lancet 1986ii:1300. 4. Renckens AUM, Jansen MJH, Van Munster PJJ, et al. Nephelometry of the kappa/lambda light-chain ratio in serum of normal and diseasedchildren. Clin Chem 1986;32:2147-9. 5. Keen H, ChiouverakisC. An immunoassay method for urinary albuminat low concentration.Lancet 1963;ii:9 13-S. 6. Feldt-RasmussenB, Dinesen B, Deckert M. Enzyme improved determination of urinary albumin in diabetics with incipient nephropathy. Scand J Clin Lab Invest 1985:45;53944.

7. Watts GF, Bennett JE, Rowe DJ, et al. Assessment of immunochemical methods for determining low concentrations of albumin in urine. Clin Chem 1986;32;1544-8. 8. Townsend JC, Sadler WA, Shanks GM. The effect of storage on the precipitation of proteins in deep frozen urine samples. Ann Clin Biochem 1987;24:111-2. 9. Erman A, Rabinow M, Rosenfeld J. Albumin determination in frozen urines-underestimated 1988;174:255-62.





CLIN. CHEM. 35/2, 310-312 (1989)

Concentrations of Carotenoids,Retinol,and Tocopherolin Plasma, in Response to Ingestionof a Meal Ellen D. Brown,AliceRose,NealCraft, KarenE.Seldel, and J. C. Smith, Jr.1 Field studies and epidemiological surveys may necessitate obtaining a blood sample from a nonfasted subject for nutritional assessments. We measured the effect of a standardized test meal, eaten after an overnight fast, on the concentrations of seven carotenoid fractions, retinol, and tocopherol in plasma of eight healthy adults. The 790-calorie test meal did not alter the measured concentrations. We conclude that blood sampled up to 4 h after breakfast can be validly used for these measurements. Recent interest in the role of carotenoids, retinoids, and tocopherol in cancer prevention has stimulated a number of

U.S. Department of Agriculture, Agricultural Research Service, Beltaville Human Nutrition Research Center, Beltsville, MD 20705. ‘Address correspondence to this author. Received September 15, 1988; accepted November 29, 1988. 310

CLINICAL CHEMISTRY, Vol. 35, No. 2, 1989

large surveys. One aspect that must be considered in determining concentrations of these ana.lytes in the blood is the effect of food ingestion. Samples collected during the fasted state are ideal, but it is more convenient to obtain blood throughout the morning from subjects, whether or not they have eaten a breakfast. Furthermore, when studiesare compared,

there are often differences in the times of blood

collection and the fasting state of subjects. Thus, the influence of a meal on these analytesbecomes a consideration in the design and interpretation of many investigations. High-performance liquid-chromatographic (HPLC) methods to measure retinol and tocopherol (1) and individual carotenoids (2) more sensitively and specifically have been reported from this laboratory. Here we have used the new methods to determine whether ingestion of a meal afteran overnight fast affects the concentration of thesenutrients in plasma. We also measured triglyceride and cholesterol concentrations in plasma as common indicators of lipid metabolism.

MaterIals and Methods Eight healthy adults (both sexes, ages 20-54 y) consumed a high-fat meal after an overnight fast. The meal consisted of the following items: 56 g of Swiss cheese, 80 g of raisin bread, 20 g of margarine, 168 g of pineapple juice, and 224 g of vanilla ice cream. Blood was collected before, and then 1 and 4 h after the meal, in all-plastic disposable syringes (Sarstedt, Princeton, NJ) with 4.5 USP units of sodium heparin (Sigma Chemical Co., St. Louis, MO) per milliliter of blood as an anticoagulant. The plasma was stored briefly at -20 #{176}C, then at -70 #{176}C. Plasma cholesterol and triglyceride concentrations were analyzed with a CentrifiChem centrifugal analyzer (Baker Instruments Corp., Allentown, PA) and Baker reagents. We measured retinol, tocopherol, and carotenoid concentrations by HPLC, using a modification of the methods of Bieri et al. (1,2), as described by Craft etal.(3). Blood samples were protected from direct sunlight, and HPLC analysis was conducted in a room illuminated with yellow light. All data were analyzed by analysis of variance and the Student-Newman-Kuels Multiple Range Test (4). Results Table 1 shows our results before (basal) and afterthe subjects had ingested the test meal. The mean triglyceride concentration in plasma was significantly higher 4 h after eating as compared with concentrations at 0 and 1 h. Cholesterol, retinol, and tocopherol concentrations did not

differ significantly among the three time periods. Table 2 presents the data for the various plasma carotenoids. Concentrations of each carotenoid and of the total carotenoids remained essentially unchanged during the experimental period.

DiscussIon We purposely formulated a test meal that contained substantial calories and was fairly high in fat, to promote absorption of fat-soluble nutrients. However, this breakfast was intended to represent a possible meal rather than a loading dose of a particular nutrient. We calculated that it contained 790 calories, 45% from fat (5). It provided 40-50% of the Recommended Dietary Allowance of vitamin A (6) and contained 0.4 mg of carotene (7), or 20% of the average

Table 1. Basal and Postprandlal ConcentratIons (Mean ± SEM) of Various Analytes In Plasma Trlgiycerld.s Hours

0 (basal) 0.68










mmoi/L imol/L 0.12 4.42 ± 0.31 1.89 ± 0.10 22.87 ± 1.74 0.15 4.63 ± 0.31 1.95 ± 0.10 23.22 ± 1.56 0.198 4.68 ± 0.36 1.95 ± 0.17 23.01 ± 1.65

Significantly different (P 0.05).

intake of total carotenoids (8). Reliable dietary tocopherol estimates were not available. As expected, plasma triglycerides significantly increased 4 h after ingestion, whereas cholesterol concentration did not change significantly. Neither retinol nor tocopherol measured by HPLC differed significantly among the three time periods (Table 1). Using a spectrophotometric method, Mejia et al. (9,10) also noted no breakfast-induced change in concentrations of serum retinol in adults or children. In the present study we examined seven separate carotenoid fractions. Neither the individual analyte concentrations nor their total changed significantly during the 4-h postprandial period. Earlier studies, in which spectrophotometric methods (9, 10) were used, measured only total carotenoids in serum and showed no change in concentration up to 4 h after a breakfast. Measurement of individual plasma carotenoids, carotenoid fractions, or both, has become increasingly important. Peto (11) speculated that a single carotenoid, beta-carotene, may be protective against cancer. Others have suggested (12, 13) that other carotenoids without vitamin A activity may be responsible for the association between consumption of greenand yellow vegetables and decreased cancer mortality. Colditz et al. (12) demonstrated a protective effectfor strawberries and tomatoes,although strawberries are relatively low in beta-carotene (F. Khachik, unpublished data, 1988), and lycopene is the major carotenoid found in tomatoes (14). Lutein and its isomers, which possess little vitamin A activity, predominate in many green and yellow vegetables (15). Presumably, in future epidemiological studies or clinical trials, these particular plasma carotenoids will be considered, not justthe total of all carotenoids. Thus, with more-sensitive HPLC methods we can conclude that the concentrations of retinol, tocopherol, and individual and totalcarotenoids do not change significantly for up to 4 h aftera hearty breakfast. Thisobservation may be useful for field studies where preprandial samples are difficult to obtain. Supported in partby a reimbursable agreement, no. Y01-CN30609, with the Division of Cancer Prevention and Control, National Cancer Institute, Bethesda, MD


References 1. Bieri JG, Tolliver TJ, Catignani GL. Simultaneous determination of aipha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am J Clin Nutr 1979;32:2143-9. 2. Bieri JG, Brown ED, Smith Jr JC. Determination of individual carotenoids in human plasma by high performance liquid chromatography. J Liq Chromatogr 1985;8:473-84. 3. Craft NE, Brown ED, Smith Jr JC. Effects of storage and handling conditions on concentrations of individual carotenoids, retinol, and tocopherol in plasma. Clin Chem 1988;34:44-8. 4. Helwig JT, Council KA, eds. SAS user’s guide. Casy, NC: SAS Institute, Inc., 1982. 5. Anon. Primary data set for USDA food consumption


Table 2. Basal and Postprandlal ConcentratIons of Carotenolds In Plasma (Mean ± SEM) Zsaxanthin/ lutein

Hours 0(basal)


1 4

634±76 616±53












246±53 246±53 229±53

112±19 112±19 112±19

417±72 417±72 399±72

1010±131 1010±131 951±112

149±37 149±37 131±37

914±168 1010±149 840±149

3420±364 3460±400 3230±364

No significantdifferences (P >0.05)withinanycolumn.

CLINICALCHEMISTRY, Vol. 35, No. 2, 1989 311

Release No. 1. Hyattaville,

MD: Human



Service, USDA, 1980. 6. National Academy of SciencesfNational Research Council, Recominended Dietary Allowances, 9th ed. Washington, DC: National Academy of Science8, 1980. 7. Food Table Version 11. Minneapolis, MN: Nutrition Coding Center, University of Minnesota, 1985. 8. Witachi JC, Houser HB, Littell AS. Prefo#{241}ned vitamin A, carotene and total vitamin A activity in usual adult diets. J Am

Diet Assoc 1970;57:13-6. 9. Mejia LA, Arroyave G. Determination of vitamin A in blood. Some practical considerations on the time of collection of the specimens and the stability of the vitamin. Am J Cliii Nutr 1983;37:147-51.

10. Mejia LA, Pineda 0, Noriega JF, Benitez J, Falla G. Significance of poetprandial blood concentrations of retinol, retinol-bind-

ing protein, and carotenoids when assessing the vitamin A status of children. Am J Clin Nutr 1984;39:62-5. 11. Pete R. Can dietary beta-carotene materially reduce human cancer rates? Nature (London) 198 1;290:201-8. 12. Colditz GA, Branch LG, Lipnick RJ, eta!. Increased green and yellow vegetable intake and lowered cancer deaths in an elderly population. Am J Clin Nutr 1985;41:32.-.6. 13. Olson JA. Carotenoids, vitamin A and cancer. J Nutr 1986;116:1127-30. 14. Zakaria M, Simpson K, Brown PR, Krstulovic A. Use of reversed-phase high-performance liquid chromatographic analysis for determination of provitamin A carotenes in tomatoes. J Chromatogr 1979;176:109-17. 15. Beecher GR, Khachik F. Evaluation of vitamin A and carotenoid data in food composition tables. J Natl Cancer Just


CLIN. CHEM. 35/2, 31 2-314 (1989)

Assessment of Renal Functionby InulinClearance: Comparisonwith CreatinineClearance as Determinedby Enzymatic Methods Fred S. Apple,’ Peter Benson,1 Paul A. Abraham,1 Thomas G. Roeano,2 and Charles E. Halstenso& We compared creatinine clearances determined by enzymatic (Kodak Ektachem 700 single-slide, Boehringer Mannheim creatinine PAP) and nonenzymatic (Jaff#{233}, HPLC) methods with glomerular filtration rate measured by inulin clearance in patients with varying degrees of renal function. The Kodak enzymatic assay gave values for creatinine 2 to 3 mg/L higher than the other methods. This resulted in significantly lower creatinine clearances than inulin clearances and creatinine clearances determined by the other methods. However, correlations between all methods for serum and unnary creatinine values and clearances were good. To avoid between-assay (enzymatic vs nonenzymatic) discrepancies, manufacturers should agree to an acceptable standard of calibration under the usual conditions used with patients. Additional Keyphrases: multilayer film analysis matographicand calorimetricassayscompared



Measurement of creatinine in serum and urine is essential in evaluation of renal function (1). Values for creatinine in serum can be used to directlyestimatethe glomerular filtration rate (GFR). However, serum creatiine is not usually measurably increased until there is a >50% lossof renal function (2). Therefore, creatinine clearance calculated from measured creatinine concentrations in serum and urine more sensitively indicates early renal dysfunction. Although inulin clearance is considered the “gold standard” for assessment of GFR, the clearance of endogenous creatinine is widely accepted(3). Laboratory evaluation of GFR depends on accurate measurement of creatinine in serum

‘Hennepin County Medical Center, 701 Park Avenue S., Minneapolis, MN 55415. 2Albany Medical Center, Albany, NY 12208. Presented in part at the 1986 and 1988 AACC national meetings, Chicago and New Orleans. Received September 26, 1988; accepted November 28, 1988.

312 CLINICALCHEMISTRY, Vol. 35, No. 2, 1989

and urine. Creatinine has traditionally been measured by variations of the alkaline picrate method, which was first described by Jaff#{233} more than 100 years ago (4). However, numerous compounds, including ketoacids, cephalosporins, and bilirubin, interfere with the Jaff#{233} reaction. Enzymatic methods for creatinine determination that are relatively free from common interferences have recently been introduced into the clinical laboratory (5). However, an earlier report(6)indicatedthat creatinine clearance as measured by enzymatic methods appeared to overestimate GFR, owing to significantly lower values for serum creatinine in comparison with the Jaif#{233} methods. To clam these issues, we have compared the clearance of creatinine as determined by the enzymatic and Jaff#{233} methods with the measurement of GFR by standard inulin clearance in patients with variousdegrees of renal function.

Materials and Methods Patients. Twenty-four patients with various degrees of renal function were selected so as to provide sixpatients in each of the following groups: group A, creatinine clearance >100 mL/min; group B, creatinine clearance 50-100 ml.) mm; group C, creatinine clearance 25-49 mL/min; and group D, creatiine clearance

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