zinc utilization in pregnant and nonpregnant women fed controlled

Zinc Utilization in Pregnant and Nonpregnant WomenFed Controlled Diets Providing the Zinc RDA1

CHRISTINE A. SWANSON2 ANDJANET C. KING3

Department of Nutritional Sciences, University of California,Berkeley, CA 94720

ABSTRACT Eight pregnant (P) women in their third trimester and 10 nonpregnant(NP) women participated in a 21-day confined metabolic study. The participants werefed semipurified liquid diets providing 20 mg zinc/day. Apparent zinc balance wascalculated and parameters of zinc status assessed. Even though an adequate amount ofa readily available form of zinc was fed, the P women consistently maintained lowermean serum zinc concentrations than the NP controls (69 jig/dl vs. 97 /Â¿g/dl).Althoughthe dietry zinc intake was constant throughout the study, most subjects' salivary zinc

levels varied greatly. In this study, salivary zinc concentration was not a sensitive indicator of zinc intake. There were no differences in the concentration of zinc in hairbetween the P and NP groups; mean values ranged from 180-220 ppm. In both groups,approximately 95% of the measured zince output was lost in the feces and only 5%excreted in the urine. The P women tended to excrete more zinc in their urine thanthe NP women (900 /ig/day vs. 650 //g/day), but they excreted less zinc in their feces.The P women demonstrated a greater apparent zinc retention than the NP women (1.9Â±0.6 vs. 0.9 Â±0.5 mg/day). This difference, however, was not statistically significant(P > 0.05). J. Nutr. 112. 697-707, 1982.

INDEXING KEY WORDS zinc â€¢zinc status â€¢zinc excretion â€¢zinc absorption â€¢human balance study â€¢pregnancy

Infertility, fetal growth retardation, ab- The concentration of zinc in serum or plasmanormal fetal development, and prolonged la- is the most commonly reported single indexbor have been observed in experimental an- of zinc nutriture. Hypozincemia associatedimals as a consequence of zinc deprivation with human pregnancy has been reported by(1-4). Parallels have been drawn between several investigators (6-10) but Jameson (6)observations in animals and conditions seen was the first to associate the phenomenon toin humans. Observations of pregnancy out- poor pregnancy outcome. Low hair zinc lev-comes of women with acrodermatitis en- els have been seen in association with zincteropathica, a congenital zinc-deficiency state, deficiency states (11-13). Hambidge andsuggest that the human fetus may be suscep- Droegemueller (10) reported that a group oftibie to the teratogenic effects of gestational pregnant women exhibited a decrease in hairzinc deficiency (5). Jameson (6) in a study zinc concentration over the course of preg-of 84 healthy pregnant women, related in- nancy. It is not known if the fall in hair zinccreased incidence of prematurity, sponta- concentration is physiological or indicativeneous abortion, congenital malformations, of marginal zinc nutriture. Saliva has beenand prolonged labor to low maternal serum suggested as a potentially useful index of zinczinc levels at the 14th week of pregnancy.

It ÃŒSnot known what proportion of preg- (eÂ©^^|neric.n Instituteof Nutrition Receivedfor publÂ¡c..ion21 Sep-

WOmen are in marginal Zince StatUS be- ' Research supported by National Institutes of Health. NIH AM 10202CaUSe Standards for aSSeSSing Zinc StatUS Of 'râ„¢â„¢> Â«MÂ«Â»USUA. Vitamin and Mineral Nutrition Laboratory.

0 Bellsville. MD 20770pregnant women have not been established. Â»TOwhomrequestsforreprintsshouldbesent

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698 SWANSON AND KING

status (14, 15). A comparison of salivary zincconcentrations of pregnant and nonpregnantwomen has not previously been made.

Measurements of zinc retention duringpregnancy are helpful for purposes of estimating zinc requirements of pregnant womenand subsequently to determine more precisely dietary zinc recommendations. Whilethe nonpregnant adult is assumed to be inzinc balance, the maternal-fetal unit shouldretain zinc during the course of pregnancy.Based on estimates of the accretion of leanbody tissue and on estimates of the zinc concentration of the tissue deposited, Sandstead(16, 17) calculated that approximately 1 mgof zinc is retained per day during the lasthalf of pregnancy. These theoretical estimates of zinc retention were used when theNational Research Council (NRC) formulated its zinc recommendation for pregnancy(18). The 20 mg/day dietary recommendation is not based on a direct measure of zincretention by pregnant women and the adequacy of the recommendation has not beenevaluated under controlled conditions.

The objectives of this investigation were1) to determine if the zinc RDA for pregnancy would maintain zinc balance in non-pregnant women and support zinc retentionin pregnant women, 2) to compare the utilization (absorption, excretion, retention) ofzinc by pregnant and nonpregnant women,and 3) under controlled conditions, to evaluate various parameters of zinc status.

TABLE1Subject

profileNonpregnantNumber

10Age.years' 26 Â±3Height,

cm 165 Â±5Weight,kgprepregnancyweight1day

1 of metabolic study 60 9 Â±8.9Stageof pregnancy,week?Prestudy

serum zinc, Â¡ig/df 81.7 Â±8.2Prestudyzinc intake, mg/day9 12 Â±9Pregnant826Â±6164

Â±861

Â±1072.0Â±10.531

Â±465.8Â±9.017Â±7

1Data expressedas mean Â±so. *Basedon participants' estimate. *On

day 1 of confinement, assuming a total gestation period of 40weeks. *Fasting venous sample, drawn approximately 10 days prior today 1 of the metabolic study. '' Based on 7-day food records, includes

zinc from food sources and mineral supplements.

METHODS

Subjects

Eight pregnant and 10 nonpregnantwomen volunteered to participate in a 21-day confined metabolic study. All of the subjects were non-smokers and based on datafrom medical histories and physical examinations were found to be healthy. The pregnant women were receiving prenatal careprior to their participation in the study, andthe only medications taken by these womenwere their prescribed prenatal vitamin-mineral supplements. Eight of the 10 nonpregnant women reported taking either vitaminor mineral supplements prior to the metabolic study. None of the nonpregnant womenreported using oral contraceptive agents orintrauterine devices in the 3 months prior tothe study.

The nonpregment women were nullipa-rous, ranged in age from 23-31, and wereof acceptable weight for height (table 1). Thepregnant women ranged in age from 19-37,were of acceptable weight for height, andhad reached their third trimester of pregnancy by day 1 of the metabolic study. Sevenof the pregnant women were primaparous,one was the mother of three children.

Experimental design

At the time of the physical exam (approximately 7 to 10 days prior to the metabolicstudy) a fasting venous sample was drawn fora serum zinc determination. In the interimbetween the physical exam and day 1 of themetabolic study each woman was asked tokeep a 7-day food record. These data wereused to assess each subject's prestudy zinc

status.During the metabolic study, the women

lived in a metabolic unit and consumed aconstant semisynthetic diet providing 20 mgzinc/day.4 Blood, saliva, and hair were eval

uated as indices of zinc status. Fasting venousblood samples were drawn on days 10 and21 of the metabolic study. Fasting saliva samples were collected directly from the parotidduct on days 1, 10, and 21. A hair samplewas collected on days 1 and 21.

*One nonpregnant subject left the study on day 17.

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ZINC UTILIZATION AND PREGNANCY 699

TABLE 2

Composition of daily diet

Item

Basal formulaEgg albumen'Malto-dextrins2Cornstarch3Sucrose4Vegetable oil5Vegetable shortening6CaCO37MgO7KC17Ca(H2P04)27Nad7Trace mineral solution7

ZnSCVHjOCuSO4FeCl3-6H20

Cholesterol8Biotin9B-1210Folate"

WaterVitamin supplement12Choline supplement13Trace mineral capsule14

Supplemental foodsapple juice, 12 fl oz; canned peaches

beef broth powder, 4 g; NaCl, 1cookies, 5 g; instant tea, 4 g.

Supplemental protein formula15Supplemental energy formula16

g/day

75.7351.5251.5225.7618.3052.161.290.591.893.750.11

.0494

.0125

.1450.300.00020.0000020.0003

317.384 capsules/day4 capsules/day3 capsules/day

, 10 oz can;g; dietetic

variablevariable

1Seymour Foods, Inc., Topeka, KS. 2Fro-Dex 42,American Maize Products Co., Buffalo Grove,IL. 3Buffalo 3401, CPC International, EnglewoodCliffs, NJ. 4Pure cane granulated, C & H Sugar Co.,San Francisco, CA. 5Cottonseed oil, Monarch Institution Foods, Doraville, GA. 6Sweetez, Proctor andGamble, Cincinnati, OH. 7Mallinckrodt ChemicalWorks, St. Louis, MO. 'Courtesy of R. Ostwald, UCBerkeley. 9 Cal Biochemicals, San Diego,CA. 10Nutritional Biochemicals Corp., Cleveland,OH. " Sigma Chemical Company, St. Louis,MO. " Miles Laboratories, Elkhart, IN. Total dailyintake, in mg: thiamin 2.0, riboflavin 3.0, pyridoxine 5.0,Ã•/-I>iotm 0.05, phytylmenaquinone 1.0, ascorbic acid50.0, folacin 0.5, niacinamide 20.0, d-calcium panto-thenate 10.0; in III: a//-rac-Â«-tocopheryl acetate 35.0,retinyl acetate 4000. 13NuLife, Gides, Inc., 250 mgcholine/capsule. 14Stynor Co., Berkeley, CA. Totaldaily intake of trace elements, in mg: manganese 4.9,molybdenum 0.24, chromium 0.5, aluminum 4.8, iodine0.15, fluoride 1.0, selenium 0.011. 1SMixture of egg

albumin and water formulated to supply 41.4 mg N/gsolution. l6Mixture of cornstarch, dextrimaltose, sucrose, cottonseed oil and water formulated to supply 2kcal/g.

The first 6 days of the 21-day study servedas an equilibration period for the balancestudies. During the last 15 days, all feces andurine were collected daily and zinc balancewas calculated. Sweat losses were not measured, but attempts were made to preventexcessive sweating and to reduce individualvariation in sweat loss. The women were notallowed to sunbathe, excercise was limitedto 30 minutes of treadmill walking per day,and the temperature of the metabolic unitwas maintained between 18 and 21Â°.

At approximately 3 months postpartum,the pregnant women and nonpregnant controls returned to the metabolic unit for a post-partum/poststudy follow-up. A fasting venous blood sample was collected during thisvisit.

Informed consent was obtained from eachsubject according to guidelines established bythe Berkeley Campus Committee on the Useand Protection of Human Subjects.

Prestudy diet records

Each women was instructed to keep a record of all food items eaten over a consecutive7-day period. Forms provided for recordkeeping listed food items in categories (e.g.,dairy products, meat, fruit, vegetables), andserving sizes were described in householdmeasures. Subjects having difficulty estimating household measures were instructed touse measuring cups and spoons until theybecame familar with estimating serving sizes.The diet records were coded and analyzedby computer. The data base of the diet analysis was a modified nutrient guide.5 The par

ticipants also provided a separate record ofany vitamin and mineral supplements takenduring the 7-day recording period.

Experiment diet

The experimental diet provided recommended intakes of all known nutrients; NRCrecommendations (18) were used as standards. The diet (table 2) consisted of a semi-purified basal formula, vitamin and mineralsupplements, supplemental protein and en-

5 Pennington. J A (1976) Dietary nutrient guide Avi Publishing Co.,

Westport. CT.

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ergy formulas, and a limited number ofwhole food items. Each woman received thesame amount of basal formula, vitamin andmineral supplements, and supplemental fooditems. The amount of supplemental proteinand energy formula fed was determined onan individual basis according to body size.In order to maintain body weight of the non-pregnant women and allow for adequateweight gain of the pregnant women, 36 kcal/kg body weight was provided. Each subjectreceived 0.8 g protein per kilogram idealnonpregnant body weight plus an additional30 grams.

A trace mineral solution added to the basalformula provided 20 mg zinc (as zinc sulfate)per day. All other dietary components contributed approximately 0.1 mg to the totaldaily zinc intake. Each day's dietary allot

ment was divided into 4 weighed feedings,which were served at 0830, 1230, 1630 and2030 hours.

Deionized water was allowed ad libitumand each subject was asked to consume atleast 1 liter/day. A raffionose-cellulose mixture (1 part raffinose:2 parts cellulose) wasadded to the basal formula to promote a morecontinuous flow of fecal material. Initiallyeach subject received 2 grams. One non-pregnant subject required 4 grams.

Collection of samples

All diet, fecal and urine samples were collected and stored in plastic containers. Thesecontainers had been washed in a Haemosolsolution, rinsed with deionized water, soakedovernight in a detergent (Radiacwash, AtomicProducts Corp., Certer Moriches, NY) containing a zinc-chelating agent, rinsed indeionized water, soaked in a 0.1 N HCl solution to remove detergent residue and thenrinsed thoroughly in deionized water.Glassware for the ashing procedure waswashed in the same manner. When the zinccontent of a sample was expected to be extremely low (ppb range) an acid-washingprocedure was followed. For example, testtubes in which saliva samples were collectedwere soaked at least 3 hours in 6 N HCl andthen rinsed with deionized water.

Dry basal diet was prepared in a stainless-

steel mixer, weighed into plastic containersand hydrated as needed during the metabolicstudy. Six diet composites were preparedwhile the metabolic study was in progress.Each diet composite included the daily allotment of the basal formula, supplementalfoods, and vitamin and mineral tablets. Thesupplemental protein and energy formulaswere analyzed separately.

Feces and urine were collected directlyinto tared plastic containers. Three-day fecalcomposites were collected and then homogenized with weighed amounts of deionizedwater. Twenty-four-hour urine collectionswere made daily, diluted to the nearest kilogram weight with deionized water, and adrop of octanol added to prevent foaming.Three-day urine composites were preparedand then frozen in plastic bottles until analyzed.

Fasting venous blood samples were drawnin acid-washed syringes fitted with 20-gaugestainless-steel needles. The samples collectedat the physical and at the poststudy evaluation were collected before 1000 hours at least10 hours after the last meal. The samplescollected during the metabolic study weredrawn between 0800 and 0830 hours, beforethe breakfast meal.

For the collection of saliva, each subjectrinsed her mouth with deionized water anda plastic collecting cup was applied to theStenson's duct of the parotid gland and held

in place by suction. Saliva flow was stimulated by having the subject place a thin sliceof fresh lemon in her mouth. Saliva passedfrom the duct through acid-washed plastictubing into acid-washed plastic tubes. Approximately 6 ml of saliva was collected in10-15 minutes. All collections were madebefore the breakfast meal between 0800 and0830 hours.

On day 1 of the metabolic study, after hairhad been shampooed and rinsed, an area approximately 2 cm square was isolated in theoccipital region of the head. Using stainlesssteel scissors, the section of hair was cut aboutl'/2 inches from the scalp. The l1^ inch lengthwas then cut to the scalp and placed in anenvelope. On day 21 the regrowth from thissection was collected. Prior to ashing, hairsamples were washed a second time to re-

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move any oils and contaminants. A methodreported by Hambidge (19) was followed.

Analysis

Prior to zinc analysis, duplicate sampleswere weighed into glass crystallization dishesand ashed overnight in a low-temperatureasher (Model LTA-604, International PlasmaCorp., Hayward, CA). Ashed samples werediluted in a 1 N HCl solution of high purity(Ultrex, J. T. Baker Chemical, Co., Phillips-burg, NJ). With the exception of saliva, samples were analyzed for zinc using flameatomic absorption spectrophotometry (Per-kin Elmer Model 306 fitted with a 3-slotburner head, Perkin Elmer Co., MountainView, CA). Aqueous working standards containing beween 0 and 1 Mg/m' were preparedas needed from a stock standard solution(Zinc reference standard, 1000 ppm, FisherScientific Co., Fairlawn, NJ). Standards wereprepared in the high purity acid and deion-ized water. Integrated 3-second readingswere made, and the zinc content of the samples determined from the standard curve byregression lines. If the zinc concentration ofa sample was greater than 1 Mg/ml it wasdiluted with l N HCl. Samples containingless than 0.06 Mg/ml were reprocessed anda more concentrated sample prepared. Samples were reprocessed if duplicate values varied by more than 5%. Reference ashes wererun daily. Bovine liver standard obtainedfrom the National Bureau of Standards (NBS)was processed and analyzed for zinc contentin the same manner as other samples. Theanalyzed zinc content of 26 NBS bovine liverstandard samples was 136 Â±4 ppm as compared to an NBS certified zinc value of 130ppm. In addition, either a pooled fecal orurine ash prepared by our laboratory was alsorun each time samples were analyzed.

Saliva samples were analyzed by anotherlaboratory.6 Saliva was prepared for zinc

analysis following the ashing procedure previously described, and zinc was determinedusing an emission spectrochemical techniquewith a silver argon arc (20).

Total nitrogen in diet, urine, and feces wasanalyzed by the micro-Kjeldahl techniqueusing a selenium catalyst (21). All samples

were analyzed in duplicate and repeated ifduplicates varied by more than 1%.

StatisticsEach subject's zinc balance was measured

once during the metabolic study (one 15-daybalance period). Zinc balance of the pregnantwomen was compared to that of the non-pregnant women by an unpaired i-test (22).For parameters in which repeated measureswere made (e.g., urinary zinc excretion wasmeasured 7 times in each subject during each3-week study) a repeated measures analysisof variance and covariance was performed(23). Unless otherwise stated, all data wereanalyzed for statistical significance at the 5%significance level (P < 0.05).

RESULTS

Parameters of zinc status

The diet records indicated that the meanprestudy zinc intake of the nonpregnant andpregnant women was 12 and 17 mg/day,respectively. Even though the pregnantwomen reported higher prestudy zinc intakesthan the nonpregnant women, they had prestudy serum zinc values that were approximately 20% lower than those of the non-pregnant women (table 1). During themetabolic study, both groups received 20 mgzinc/day and the pregnant women continuedto maintain serum zinc values which weresignificantly (P < 0.05) lower than those ofthe nonpregnant women (table 3). The average values for the pregnant and nonpregnant women, respectively, were 70 and 97Mg/dl.

The nonpregnant women demonstrated asignificant increase in serum zinc from thetime of the physical (82 Mg/dl) to day 21 (99Mg/dl). The values of the pregnant womenwere relatively constant during this time period (66 vs. 68 Mg/dl). The range of observedvalues for the pregnant and nonpregnantwomen, respectively, were 48-78 Mg/dl and72-115 Mg/dl. At the time of the poststudy/postpartum evaluation the serum zinc con-

â€¢Courtesy of Dr K. Michael Hambidge, University of Colorado Medical

Center. Denver. CO 80262. NIAMDD. Grant No. 5 R22 AM 12432.

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TABLE 3

Comparison of fasting serum zinc values ofnonpregnant and pregnant women1

TimePrest

udy2Day

10Day

21Poststudy3NonpregnantHg/dl81.7

Â±2.6'(10)95.4

Â±3.8'(10)98.8

Â±3.91(10)88.8

Â±5.7'(7)Pregnantng/dl65.8

Â±3.2b(8)71.1

Â±2.1b(8)68.0

Â±1.7b(8)82.0

Â±3.2"(8)

1Values shown are the mean Â±SEM,number of subjects in parentheses. 2Approximately 7-14 days priorto the metabolic study. 'Approximately 3 monthspostpartum and 3 months postmetabolic study for theP & NP women, respectively. *jl>Values in same rowwith different superscripts are significantly different (P< 0.05).

centrations of the two groups were not significantly different, 89 versus 82 Mg/dl.

During the metabolic study the pregnant

i.iOr

1.00

.80

.60

Nonpregnant

3 6 9 12 15 18 21TIME (days)

Fig. 1 Urinary zinc excretion of pregnant (n = 10)and nonpregnant women (n = 8) fed 20 mg of zinc (aszinc sulfate) per day. Points and vertical bars representmeans Â±SEM.

women had a greater mean 24-hour urinaryzinc output than the nonpregnant women(fig. 1). The average excretion of the pregnant women was 900 Â¿ig/daycompared toan average output of 650 /ig/day for the non-pregnant women. The difference betweenthe two groups, however, was not statisticallysignificant at any point in time (P > 0.05).Individual values ranged from 260 to 1600jÂ«g/day.

By day 12, each individual subject maintained a relatively constant urinary zinc output, but the variation between subjects remained large. For example, one nonpregnantsubject maintained a mean output of 560Â±40 Mg/24 hours, whereas another maintained a mean excretion of 1090 Â± 100/ig/day.

The nonpregnant women demonstrated a60% increase in urinary zinc excretion duringthe metabolic study. The change was significantly correlated to the increase in serumzinc (P < 0.05). The urinary zinc excretionof the pregnant women increased only 15%and this change was not statistically significant (P Ã¨0.05).

Fasting saliva samples were drawn on days1, 10, and 21 of the metabolic study and atthe poststudy evaluation (table 4). The valuesof the pregnant women were not statisticallydifferent from those of the nonpregnantwomen and there were no significant changes

TABLE4Fasting salivary zinc values of nonpregnant

and pregnant women1*

Time Nonpregnant Pregnant

Day1Day

10Day

21Poststudy2ppb51

Â±6.3'(9)46

Â±8.2-(9)38

Â±9.5'(9)56

Â±13.1*(6)ppb38

Â±5.8b(9)49

Â±9.5'(8)44

Â±8.T(8)50

Â±8.7'(7)

' Values shown are the mean Â±SEM,number of subjects in parentheses. 2Approximately 3 months postpartum and 3 months postmetabolic study for the pregnant and nonpregnant women, respectively. *'bValuesin same row with different superscripts are significantlydifferent (P < 0.05).

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in saliva zinc concentration as a function oftime in either group. During the metabolicstudy when zinc intake was constant (20 mg/day) the mean saliva values for both groupsranged from 38 to 51 ppb. However, as indicated by the SEM, a relatively large estimate of variance was associated with eachmean value. As an example, the values forone nonpregnant subject were 86 and 32 ppbon days 1 and 21, respectively; while thoseof another nonpregnant woman were 40 and100 ppb on the same days. Five of the subjects had salivary zinc values in the 10-20ppb range on at least 1 day of the metabolicstudy.

The pregnant women had a mean hair zincconcentration of 192 Â±6 and 202 Â±11 fig/g (mean Â±SEM,n â€”7) on days 1 and 21, respectively. The corresponding data for thenonpregnant women were 186 Â±8 and 183Â±16 /ig/g (n = 9). The difference betweenthe pregnant and nonpregnant women wasnot statisically significant. Some of the subjects objected to the hair-cutting procedureand did not provide a hair sample.

Zinc balance

Fecal zinc excretion accounted for 95% ofthe measured zinc loss in both the pregnantand nonpregnant women (table 5), valuesranged from 92 to 97%. Urinary zinc excretion accounted for the remaining 5%, valuesranged from 2 to 8%. The pregnant womenlost less fecal zinc than the nonpregnant

TABLE 5

Zinc excretion, apparent absorption, and retention bypregnant and nonpregnant women12

Zinc intake,mg/dayFecalzinc,mg/dayUrinary

zinc,mg/dayApparent

retention,mg/dayApparent

absorption,%Nonpregnant

<n =9)20.1

Â±0.118.3Â±1.20.7

Â±0.21.1

Â±1.39.0

Â±6.5Pregnant(n

-8)20.1

Â±0.016.8Â±1.60.9

Â±0.32.4

Â±1.616

4 Â±7.8Statistical

significanceNS"NSNSNSNS

1Data expressed as mean Â±SD. *Measurements made over the last 15days of a 21-day confined metabolic study in which a constant diet providing20 mg zinc/day was fed. 'NS. not statistically significant. (P

> 0 05)

ICT>

J

ÃœJU

mozN

6.0

5.0

4.0

3.0

2.0

..o

I-1.0

A Pregnant

â€¢Nonpregnant

â€¢ AÂ»

A

1.0I

2.0 3.0 4.0

-1.0 L

NITROGEN BALANCE (g/day)

Fig. 2 Relationship between nitrogen and zinc retention in pregnant (A) and nonpregnant (â€¢)women.Nitrogen and zinc balance measured over the last 15days of a confined metabolic study in which a constantformula diet was fed.

women, resulting in higher apparent absorption values, 16% versus 9%. The differencebetween the two groups was not statisticallysignficant at the 5% level (P = 0.09).

The pregnant women had a higher apparent retention of zinc than the nonpregnant women, 2.4 versus 1.1 mg/day. Threeof the nonpregnant women were in slightlynegative zinc balance (approximately â€”0.5mg/day); values ranged from â€”0.6to +2.5mg/day. All the pregnant women were inpositive balance retaining at least 0.5mg/day; values ranged from +0.5 to +5.5mg/day.

The relationship between zinc retentionand the deposition of lean body mass wasevaluated by correlating zinc balance to nitrogen balance (fig. 2). The pregnant womenretained significantly more nitrogen (P< 0.05) than the nonpregnant women (1.9 vs.0.8 mg/day), and as described above theyhad a higher mean retention of zinc. However, when each subject's nitrogen data andzinc data were paired there was no signifi-

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cant correlation between the two parameters(r = 0.13, P > 0.05).

Pregnancy outcome

All of the pregnant women gave birth tonormal healthy full-term infants. Averagebirth weight and lengths were within thenormal range of healthy newborns. Weightgain of all but one participant exceeded therecommended optimum of 12.5 kg.

DISCUSSION

Parameters of zinc status

Prior to the metabolic study, the serumzinc values of the pregnant women were 20%less than those of the nonpregnant controls.This difference was exaggerated during themetabolic study because the nonpregnantwomen demonstrated a significant increasein serum zinc, while the pregnant womenmaintained relatively constant concentrations. That the nonpregnant women demonstrated a significant increase in serum zincfrom the time of the physical to the end ofthe metabolic study is likely the result of theirzinc intake increasing from about 12 mg/dayto 20 mg/day. The serum values of the pregnant women are more difficult to interpret.Any addition of zinc to the total circulatingplasma pool may have been masked by he-modilution; expansion of maternal blood volume is reported to increase through the 34thweek of pregnancy (24). Furthermore, utilization of zinc by the fetus may also serveto depress maternal concentrations. Nonetheless, the pregnant women did consistentlymaintain lower zinc concentrations than theirnonpregnant counterparts.

Our data do not agree with the findingsof Jameson (6) who has associated the hy-pozincemia of human pregnancy with a zincdeficiency syndrome. He has reported (25)that healthy women who undergo uncomplicated pregnancies and deliver normal infants show only a slight decrease in serumzinc levels throughout pregnancy as compared to healthy nonpregnant women. In ourinvestigation, pregnant women fed 20 mg ofa readily available form of zinc consistentlymaintained lower serum zinc concentrationsthan nonpregnant controls. We interpret

these observations to mean that the hypozin-cemia of pregnancy is physiological and thatserum values of pregnant women should notbe evaluated against those of nonpregnantwomen, but rather that new standards shouldbe developed for pregnancy. The eight pregnant women in this investigation maintainedfasting serum zinc values in 60-70 /ig/dl

range during their third trimester. A largepopulation of women should be studied inorder to determine the lower limit of thenormal range. Hambidge and Maur (26) haveprovisionally set that range at 45-50 Mi/dl.

Urinary zinc excretion has been regardedas a poor index of zinc status because bothhypo- and hyperzincuria have been observedin zinc deficiency. However, hyperzincuriahas been seen most commonly in hospitalizedpatients (27) whose metabolic abnormalitiesmay have altered urinary zinc excretion.Healthy individuals fed zinc-deficient dietsexhibit hypozincuria (27, 28). Healthy youngadult men7 and women (28) fed zinc-defi

cient diets (about 0.3 mg/day) decreasedtheir urinary zinc excretion to 100-150 fig/day or less. In these depletion studies thedecrease in urinary zinc output occurred inmost cases with a decrease in serum zinc concentration, indicating that urinary zinc output is responsive to changes in dietary intakeand that the urinary response corresponds toanother paramter of zinc status. In our study,the nonpregnant women demonstrated anincrease in serum zinc concentration and anincrease in urinary zinc output. Presumablyboth responses reflect the increased zinc intake during the metabolic study as comparedto the subject's reported prestudy intake and

furthermore indicate some renal regulationof zinc homeostasis.

Recently, saliva has been collected andanalyzed for zinc content to determine if thisfluid is sensitive to changes in zinc intake andstatus. Several investigators have found salivato reflect zinc nutriture (14, 15), but we wereunable to demonstrate such a relationship.The laboratory that performed the salivaanalysis indicated that saliva values of 10-20ppb were considered indicative of marginalzinc status. It this were true, then several of

' Baer. M. T. (1979) Experimental zinc depletion in young men. Doc

toral dissertation. University of California, Berkeley.

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our subjects were drifting in and out of marginal zinc status during the metabolic study.Considering the level and form of zinc fed,it is unlikely that this was in fact occurring.Given the tremendous amount of intra- andinterindividual variation and considering thatzinc intake was held constant throughout thestudy, parotid saliva was not a sensitive indexof zinc intake. It may be that some of thevariation in salivary zinc values could be explained by differences in salivary flow rates.Individual subjects required anywhere from5 to 25 minutes to provide a 6-ml sample.

It is difficult to know what physiologicalsignificance, if any, to attribute to the hairzinc data. Mean values of 160-180 ppm arecommonly reported for healthy individualsand values between 70-100 ppm are considered low and possibly indicative of inadequate zinc status (10, 26, 30, 31). Values forboth the pregnant and nonpregnant subjectswere within the normal range. Hambidgeand Droegemueller (10) reported that pregnant women show a decrease in hair zincconcentration from early to late pregnancy.We did not observe any change in hair zincconcentration of our pregnant women duringthe 21-day study. Possibly our collection period was too short to make such a determination. If hair was a particularly sensitiveindex of zinc intake one would have anticipated an increase in the hair zinc concentration of the nonpregnant women corresponding to their increased serum zinc concentration and to their increased urinary zincexcretion. The hair zinc concentration, however, remained constant. Admittedly oursample size was small and collection periodsbrief, but it appears that hair was not a particularly sensitive index of zinc status in ourstudy.

Zinc utilization

The pregnant women showed a tendencyto lose more zinc in their urine than the non-pregnant women. There are several possibleexplanations for the phenomenon. It may bethat renal conservation mechanisms do notoperate when a relatively high level of anavailable form of zinc is fed. Urinary histi-dine output was not measured, but histidineis a zinc chelator in vitro (32, 33), and his

tidine excretion has been reported (34, 35)to increase severalfold during pregnancy.Finally, the response may be nonspecific andrelated to the increase in the glomerular filtration rate associated with pregnancy (35).Hytten et al. (35) have linked increased urinary excretion of many water-soluble vitamins, several amino acids, and glucose tochanges in glomerular filtration rate.

Since the pregnant women excreted lesszinc in their feces than the nonpregnantwomen, they had a greater mean apparentzinc absorption. Unfortunately, the balancemethod does not distinguish between unab-sorbed dietary zinc and zinc of endogenousorigin. Therefore, it is not certain whetherthe higher mean apparent absorption valuesof the pregnant women reflect increased absorption of dietary zinc or decreased excretion of endogenous (metabolic) zinc.

Zinc retention

The zinc RDA of 20 mg when fed as zincsulfate supported an average positive balanceof zinc in both the pregnant and nonpregnantgroups. However, sweat and dermal zinclosses were not determined and can contribute significantly to zinc balance. Several investigators (36-38) have reported that approximately 1 mg of zinc is lost per liter ofsweat. Assuming that each woman in ourstudy lost 1 liter of sweat per day with anassociated zinc loss of 1 mg/day, then thenonpregnant group would have been in zincequilibrium and the pregnant group wouldshow a zinc retention of 1 mg/day. Thesedata closely approximate the theoretical estimates (16-18).

Although the mean values agree with thetheoretical estimates, closer examination ofthe data reveal certain limitations of the balance method. First, some of the individualbalance data do not seem reasonable. If oneassumes a sweat and dermal zinc loss of 1mg/day, then three nonpregnant and onepregnant women were in negative zinc balance. None of these subjects showed any clinical or biochemial manifestations of zinc depletion. Second, zinc recommendations forpregnancy are based on the assumption thatzinc is accumulated with the deposition oflean body mass. Our data show that there

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was no correlation between these two parameters when deposition of lean body mass isestimated by nitrogen retention. This is notto imply that the basis of the theoretical estimate is invalid but rather suggests that thebalance method is not sufficiently sensitiveto test the relationship experimentally. Giventhe variability in our balance data, we suggest that the data is interpreted with cautionand that the mean values be regarded onlyas estimates of zinc retention, specific to theexperimental conditions. It may be thatmuch longer balance periods are needed inorder to obtain useful balance data.

ACKNOWLEDGMENTS

The authors wish to thank Janet A. Appeland Ann Larson Wright for coordinating thestudy. The contributions of Fran Costa, Denise Rousseau, Doris Armstrong Fleck andNancy Bradfield are gratefully acknowledged. Special thanks are extended to theparticipants and their families.

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zinc utilization in pregnant and nonpregnant women fed controlled

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