Hypercalciuric Response to Dietary Supplementation with DL-Methionine and Ammonium Sulfate

Hypercalciuric Response to Dietary Supplementation with DL-Methionine and Ammonium Sulfate AMY J. LENT and ROBERT F. WIDEMAN1 Department of Poultry Science, The Pennsylvania State University, University Park, Pennsylvania 16802

1994 Poultry Science 73:63-74

when the parathyroid glands are surgically removed or when intravenous Ca Hypercalciuria and hypophosphaturia infusions are used to suppress parathyroid are consistently observed when immature function (Wideman, 1987; Clark and WideSingle Comb White Leghorn pullets are man, 1989; Wideman et al, 1989). Histofed diets containing more than 3% Ca and logical and biochemical observations furless than .45% available P (aP) (Wideman ther indicate that the parathyroid glands et al., 1985, 1989; Glahn et al., 1988a, 1989). are inhibited when immature pullets are These renal responses suggest that high dietary Ca:aP ratios inhibit the secretion of fed diets having high Ca:aP ratios (Shane parathyroid hormone, because hypercal- et al, 1969). Based on these observations, it ciuria and hypophosphaturia also occur has been proposed that low P availability may be essential for high dietary Ca to maximally inhibit the parathyroid glands (Wideman, 1987; Wideman et al, 1989). According to this hypothesis, low dietary Received for publication June 7, 1993. Accepted for publication September 1, 1993. aP stimulates the synthesis of the x To whom correspondence should be addressed. active vitamin D metabolite, 1,253 Current address: Department of Poultry Science, dihydroxycholecalciferol, which in turn University of Arkansas, Fayetteville, AR 72701. INTRODUCTION

63

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ABSTRACT Renal Ca and inorganic P (Pj) excretion were evaluated in Single Comb White Leghorn pullets reared on diets containing 1 or 3.5% Ca alone or supplemented with .6% DL-methionine or .53% ammonium sulfate. Plasma and urine samples were collected during a CONTROL period, and while 200 mM Ca was infused intravenously (Ca-LOADING). Excess Ca, whether supplied chronically in the feed or infused acutely into birds fed 1% Ca diets, significantly reduced glomerular filtration rates, effective renal plasma flow rates, and Pj excretion rates and significantly increased Ca excretion rates and urine pH. Birds fed diets supplemented with DL-methionine and ammonium sulfate maintained significantly lower plasma Ca concentrations during the CONTROL and Ca-LOADING periods than birds fed the respective 1 or 3.5% Ca basal diets. When compared with birds fed the respective 1 or 3.5% Ca basal diets, birds fed the 1% Ca diet supplemented with ammonium sulfate or the 3.5% Ca diet supplemented with DL-methionine had significantly higher absolute urinary Ca excretion rates during Ca-LOADING. Fractional Ca excretion during Ca-LOADING was significantly higher in birds fed 3.5% Ca supplemented with DL-methionine or ammonium sulfate than in birds fed the 3.5% Ca basal diet. These results indicate that DL-methionine and ammonium sulfate accelerated urinary Ca excretion and reduced Ca retention in the extracellular fluid. The hypercalciuric efficacies of DL-methionine and ammonium sulfate were revealed only when the filtered load of Ca was increased through intravenous Ca infusions. (Key words: kidney function, acid-base balance, calcium, phosphorus, pullet)

64

LENT AND WIDEMAN

nutritionally induced hyperabsorptive hypercalciuria (Wideman et al, 1989). The hypercalciuric response of domestic fowl to dietary supplementation with MHA originally was attributed to the well documented renal responses to sulfurcontaining amino acids (Wideman et al, 1989). High dietary levels of sulfurcontaining amino acids increase the excretion of sulfate anion coupled with H+ and ammonia, thereby acidifying the urine and causing hypercalciuria in mammals (Lemann and Relman, 1959; Zemel et al, 1981). Presumably the increased dietary sulfate content shifts cation:anion balance toward a more acidic value (Mongin, 1968). However, because MHA possesses inherent acidity independent of its sulfur content, the observed influences of MHA on avian urine pH and Ca excretion cannot be accepted as conclusive proof that birds and mammals respond similarly to increased intakes of sulfur-containing amino acids. Therefore, the present study was conducted to evaluate the renal responses of pullets to DL-methionine or ammonium sulfate, sulfur-containing compounds that do not possess inherent acidity. Renal function was evaluated during a control clearance period and during intravenous infusion of Ca designed to suppress parathyroid function independent of dietary Ca:aP ratios. MATERIALS AND METHODS

Female DeKalb-XL chicks obtained from a commercial hatchery were wingbanded and vaccinated for Marek's disease. They were brooded together and had ad libitum access to a commercial chick starter ration through 4 wk of age, when they were divided into six diet treatment groups with eight cage replicates per diet treatment and five birds per cage (100 cm x 102 cm floor space per cage). From 5 to 14 wk of age, they were fed diets formulated to contain: normal Ca (NC, 1% Ca); normal Ca plus .6% DL-methionine (NCDL), normal Ca plus .53% ammonium sulfate (NCAS), high Ca (HC, 3.5% Ca), high Ca plus .6% DL-methionine (HCDL), or high Ca plus .53% ammonium sulfate (HCAS). Compositions of the basal NC and HC diets have been published previ-

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accelerates intestinal absorption of both inorganic P (PJ and, unavoidably, Ca (Morrissey and Wasserman, 1971; Swaminathan et al, 1978; Norman, 1985). Either the resulting hyperabsorptive hypercalcemia or elevated plasma 1,25-dihydroxycholecalciferol concentrations, or both, can inhibit the parathyroid glands directly, leading to increased urinary Ca excretion and decreased urinary Pi excretion (Henry et al, 1977; Cantly et al., 1987; Wideman, 1987; Wideman et al, 1989). This hypothesis received further support from evidence that the aP declines as the dietary CA content increases (Shafey, 1993) and that urinary Ca concentrations and plasma 1,25-dihydroxycholecalciferol concentrations increase when laying hens are fed diets having high Ca:aP ratios (Rao and Roland, 1990; Frost et al, 1991). In addition to influencing urinary Ca and P; excretion, high dietary Ca:aP ratios consistently induce a metabolic alkalosis accompanied by a compensatory increase in urine pH (Wideman et al, 1985, 1989; Glahn et al, 1988a, 1989; Lent and Wideman, 1993). Presumably this nutritional influence on acid-base balance reflects the "alkalinizing" effect of shifting the dietary cation:anion balance (Mongin, 1968; Wideman et al, 1989), because reducing the circulating titers of parathyroid hormone should acidify rather than alkalinize the urine pH (Laverty and Wideman, 1985; Glahn et al, 1988a). In this context, it is important to note that acidosis strongly stimulates Ca excretion in immature pullets (Glahn et al, 1988a), therefore, the relative alkalinizing impact of high dietary Ca:aP ratios may partially attenuate the concurrent hypercalciuric response. For example, dietary supplementation with methionine hydroxy analog free acid (MHA), which is an inherently acidic sulfur-containing analog of methionine, significantly acidified the urine and increased both fractional and absolute Ca excretion when immature pullets reared on a high Ca diet were infused with a 100-mM Ca solution (Wideman et al, 1989). These results suggest the alkalinizing effects of diets having high Ca:aP ratios can be counteracted by MHA, resulting in a further enhancement of the

HYPERCALCIURIC RESPONSE

ethylene urine collection cannula was sealed inside the cloaca using cyanoacrylate adhesive. Throughout the surgical preparations, a solution containing 25 g/L mannitol, 1.5 g/ L inulin, and 1.5 g/L p-aminohippuric acid (PAH) was infused at a rate of .2 mL/kg body mass per min via the venous cannula. The same infusion rate was maintained throughout the experiment. Mannitol was used as a mild osmotic diuretic to obtain a consistent rate of urine flow. After surgical preparations were complete, a 30-min stabilization period elapsed, then CONTROL samples were coUected for 20 min. A second solution containing 25 g/L mannitol, 1.5 g/L inulin, 1.5 g/L PAH, 100 mM CaCl 2 , and 100 mM Ca a c e t a t e (Ca[CH3COO]2-H20) then was infused. The composition of this "Ca-LOADING" infusate was designed to provide both metabolizable (acetate) and nonmetabolizable (chloride) counterions for Ca. In the absence of metabolizable anions, CaCl2 Renal Function Protocol infusions cause metabolic acidosis and urinary acidification in mammals and birds Kidney function studies were conducted (Haldane et al, 1923; Bottje and Harrison, using eight birds per diet treatment at 14 wk 1985). The Ca-LOADING proceeded for 35 of age. One bird was selected randomly min, then urine was coUected for 40 min from each cage replicate and was anesthe- during ongoing Ca infusion. Arterial blood tized with an intramuscular injection of samples were withdrawn at the midpoints AUobarbital (Dial, 25 mg/mL, 3.5 mL/kg of the CONTROL and Ca-LOADING urine body mass).5 General surgical anesthesia coUection periods. FinaUy, birds were euwas maintained by injecting supplemental thanatized with intravenous injections of doses of aUobarbital (.5 to 1 mL) as needed. 20% urethane, their kidneys were removed, Lidocaine (2%)6 was injected intracutane- blotted to remove surface moisture, and ously as a supplemental local anesthetic at weighed. surgical incision sites. A brachial vein was cannulated for intravenous infusions, and a carotid artery was cannulated for blood Plasma and Urine Analysis sample coUection. Urine was collected as Ammonium heparin (200 U/mL) was described previously (Glahn et al, 1988a). added to blood samples to prevent clotting, Briefly, an abdominal incision was made the blood was centrifuged, and the plasma and the colon was hgated near its junction with the cloaca to prevent fecal contamina- was stored frozen at -4 C. Timed urine tion of the urine. The abdominal incision samples were coUected in preweighed was closed with wound clips and, after tubes for gravimetric determination of flushing and swabbing the cloaca, a poly- urine flow rate (UFR). Freshly collected urine was used for measurements of osmolaUty (Wescor Model 5100 C vapor pressure osmometer)7 and pH. A portion (.2 mL) of 2 each urine sample was mixed with an equal ICN Biochemicals, Cleveland, OH 44101. 3 Monsanto Agricultural Co., St. Louis, MO 63167. volume of .5 M Uthium hydroxide to free ^igma Chemical Co., St. Louis, MO 63178-9916. trapped cations (Ca2+, Na+, K+) from uric 5Ciba Pharmaceutical Co., Summit, NJ 07901. 6 Interstate Drug Exchange, Amityville, NY 11701. acid precipitates, and the dUuted samples were stored frozen at -4 C for subsequent TWescor, Inc., Logan, UT 84321.

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ously (Wideman et al, 1989). Both basal diets contained 18% crude protein, .4% aP, and 3,029 kcal ME/kg. DL-methionine (feed grade, 99%2) was blended with the basal diets to reach desired levels of .6% added methionine. This level of DLmethionine supplementation is similar to the .6% level of MHA used previously (Wideman et al, 1989). Feed analyses conducted by Monsanto Agricultural Co.3 indicated the presence of methionine at .26 to .31% in the NC, NCAS, HC, and HCAS diets (basal methionine levels) and .75 to .91% in the NCDL and HCDL diets. Ammonium sulfate ([NH4]SCV F- W. 132.13) was blended with the basal diets to reach the desired level of .53% ammonium sulfate, which on a molar basis adds sulfur equal to that contributed by .6% DLmethionine. All birds had ad libitum access to feed and water throughout the trial. Feed consumption was not measured.

65

66

LENT AND WIDEMAN

Models procedure, with infusion protocol (2 = CONTROL or Ca-LOADEMG) and diet treatment groups (NC, NCDL, NCAS, HC, HCDL, HCAS) as the main effects. For comparisons of CONTROL values obtained from all birds fed 1 vs 3.5% Ca, diet groups were pooled accordingly (n = 24 per pooled mean), and analyzed by ANOVA comparing dietary Ca content (or 3.5% Ca) as the main effect. Differences between least squares means were considered significant at P < .05 using repeated t tests. RESULTS

When compared during the CONTROL period, birds fed diets containing 3.5% Ca had significantly higher urine pH values and significantly lower values for glomerular filtration rate and effective renal plasma flow than birds fed 1% Ca diets (Table 1). Birds fed 3.5% Ca had significantly lower plasma Ca concentrations and were excreting Ca in the urine at a 15-fold higher rate than birds fed 1% Ca diets (2.1 vs .14 jiM/kg per min, respectively). The combination of low plasma Ca concentrations and low glomerular filtration rate values resulted in significantly lower filtered loads of Ca for birds fed 3.5% Ca compared with birds fed 1% Ca diets. Fractional Ca excretion was significantly elevated in birds fed 3.5% Ca diets, indicating high dietary Ca suppressed renal tubular Ca reabsorption and caused ongoing urinary Ca loss even after Ca depletion from the extracellular fluid had significantly reduced the filtered load of Ca. Dietary Ca did not alter plasma Pj concentrations during the CONTROL period, but the filtered load of Pj was significantly lower in birds fed 3.5% Ca compared with birds fed 1% Ca, primarily reflecting the influence of dietary Ca on glomerular filtration rate (Table 1). In addition to reducing the filtered load of Pj, Statistical Analysis 3.5% Ca diets also significantly stimulated Urine pH values were converted to H+ net renal tubular P; reabsorption, conseconcentrations for statistical analysis. Mean quently both absolute (EK) and fractional values for each variable were calculated (FEK) excretion rates were significantly using n = 8 birds per diet treatment. Data lower in birds fed 3.5% Ca diets than for were analyzed by ANOVA in a 2 x 6 birds fed 1% Ca diets. Dietary Ca did not factorial arrangement using the SAS® soft- alter body mass, urine flow rate, urine ware (SAS Institute, 1982) General Linear osmolality, plasma osmolality, plasma

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analyses. Colorimetric methods were used to measure urine and plasma Pj, inulin, and PAH (Fiske and Subbarow, 1925; Brun, 1957; Waugh, 1977). Sodium and K were measured by flame photometry, and Ca was measured by atomic absorption spectrophotometry. The glomerular filtration rate (GFR) was calculated as the clearance of inulin: GFR = Cln = (UFR x [InD/Mp, where [ln]u and [ln]p are the urine and plasma inulin concentrations, respectively. The fraction of filtered water excreted as urine was calculated as: FRVE = UFR/GFR, where UFR equals the excreted portion of the glomerular ultrafiltrate and GFR equals the rate at which plasma water enters the tubules by glomerular filtration. The effective renal plasma flow rate (ERPF) was calculated as the clearance of PAH: CPAH = (UFR x [PAH] u )/[PAH] p , where [PAH]U and [PAH]p are the urine and plasma PAH concentrations, respectively. The rate at which Ca enters the tubules through glomerular filtration was calculated as the filtered load of Ca: FLc, = (GFR x [Ca^), where [Ca]^ is the ultrafilterable (not bound to protein) fraction of total plasma Ca (Wideman, 1987; Wideman et al, 1989). The rate at which Ca was excreted in the urine was calculated as: ECa = (UFR x [Ca]u), where [Ca]u is the Ca concentration of the urine. The fraction of the filtered load of Ca excreted in the urine, or fractional excretion of Ca (FECa), was calculated as: FEQ, = ([Ca] u /[CaU/([ln] u /[ln] p ). This calculation normalizes absolute Ca excretion rates for possible changes in FLCa, which can occur due either to a change in GFR or to a change in [Ca]^. Filtered loads, urinary excretion rates, and fractional excretions of Pj, Na, and K were calculated as described for Ca above, except that plasma Pj, Na, and K are considered freely (100%) filterable (Wideman, 1987; Wideman et al, 1989).

67

HYPERCALCIURIC RESPONSE

TABLE 1. Plasma and kidney function values during CONTROL urine collection periods, comparing all pullets fed diets containing normal (NC: 1%) or high (HC: 3.5%) calcium (Mean ± SEM, n = 24) All NC

All HC

P

Body mass, g Urine flow rate, mL/kg per min GFR,1 mL/kg per min ERPF,2 mL/kg per min Urine pH Urine osmolality, mOsM/kg Plasma osmolality, mOsM/kg Total plasma Ca, mM Ca filtered load, /iM/kg per min Ca excretion rate, j*M/kg per min FECa3 Plasma P;,4 mM Pi filtered load, /tM/kg per min P; excretion rate, /iM/kg per min FEP(3 Plasma Na, mM Na excretion rate, j*M/kg per min FENa3 Plasma K, mM K excretion rate, /iM/kg per min FEK3

1,270

1,270

NS NS .0001 .0001 .002 NS NS .0002 .0001 .02 .0002 NS .0001 .0001 .0001 NS NS NS NS NS NS

±25 .27 ± .03 6.07 ± .36 60.6 ± 4.2 5.29 ± .06 ± 15 292 277 ± 4 3.09 ± .29 10.1 ± .95 .14 ± .03 .015 ± .002 1.49 ± .04 9.04 ± .56 5.09 ± .51 .58 ± .06 141 ± 5 6.73 ± .74 .009 ± .002 3.18 ± .08 5.85 ± 1.19 .30 ± .04

±28 .23 ± .02 4.13 ± .36 25.8 ± 2.9 5.60 ± .09 ±16 268 ±11 282 1.45 ± .16 3.37 ± .46 2.10 ± .41 .78 ± .17 1.39 ± .05 5.68 ± .46 1.12 ± .27 .18 ± .04 146 ± 3 4.36 ± .61 .008 ± .001 3.17 ± .11 4.63 ± .54 .39 ± .05

'GFR = glomerular filtration rate. ERPF = effective renal plasma flow rate. 3 Fraction of the filtered load excreted; no units. 4 P ; = inorganic P. 2

concentrations of Na and K, or the renal handling of these electrolytes. For birds fed 1 or 3.5% Ca, the mass of right kidneys (6.32 ± .20 vs 6.52 ± .22 g, respectively) and left kidneys (5.97 ± .16 vs 6.34 ± .20 g, respectively) did not differ, nor was there evidence of Ca-induced kidney damage or urolithiasis in the birds used for renal function studies. Kidney function values for the separate diet treatment groups during CONTROL and Ca-LOADING urine collection periods are shown in Table 2. For the groups fed 1% Ca diets, Ca-LOADING significantly reduced glomerular filtration rate (NC, NCDL, NCAS) and effective renal plasma flow (NC, NCDL), and significantly increased urine pH (NC, NCDL, NCAS). Significant changes in these variables were not observed during CaLOADING in the groups fed 3.5% Ca (HC, HCDL, HCAS), presumably because the 3.5% Ca diet previously had triggered similar responses (Table 1). Urine flow increased significantly only in the HC group during Ca-LOADING; however cal-

culating the fraction of filtered water excreted demonstrated a significant tubular diuresis in the NC, HCDL, and HCAS groups (Table 2). The effect of CaLOADING on the fraction of filtered water excreted can be attributed to an osmotic diuresis associated with increased urinary Ca excretion because, with the exception of the HCAS group, urine osmolality (Table 2) and free water clearance (C H 0 ; data not shown) were not significantly altered during the intravenous Ca infusion. No other consistent intergroup differences were detected among the renal function parameters compared in Table 2. During CONTROL urine collection periods for example, birds fed the NCAS diet had the highest glomerular filtration rates, whereas birds fed the NC diet had the highest effective renal plasma flow rates. Other intergroup similarities and differences tended to reflect the general responses to dietary Ca shown in Table 1. Total plasma Ca was significantly lower during CONTROL and Ca-LOADING periods, when birds fed the 1% Ca diets

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Variable

CONTROL Ca-LOADING CONTROL Ca-LOADING

CONTROL Ca-LOADING

CONTROL Ca-LOADING

CONTROL Ca-LOADING

CONTROL Ca-LOADING

Infusion protocol

.42 d

293 261 5.41 6.26

.Oic .05a

.05" .05*

± 22 ab ± 6" ± .15b ± .28a

.05 ± .19 ±

.25 ± .36 +

70.5 ± 9.8a 36.9 ± 11.2**

5.21 ± 2.62 +

NC

.01<: .Olbc

.05b .04ab

± 30* ± 5b b ± .06 ± .15"

.05 ± .11 ± 279 260 5.24 5.72

.64b .34"1

± 5.0b ± 2.3C

.27 ± .37 ±

49.2 26.4

5.74 ± 3.39 ±

NCDL .22a SI1*

304 267 5.21 5.99

.01<: .01 bc

.04b .05*

±28* ± 6b b ± .08 ± .108

.04 ± .09 ±

.29 ± .42 ±

62.0 ± 4 .iab 39.4 ± 2.31*

7.27 ± 4.77 ±

NCAS

1

Means (± SEM, n = 24) with no common superscript within a variable differ significantly (P < .05). (Urine flow rate)/(glomerular flow rate); no units.

a_d

Urine pH

Urine osmolality, mOsM/kg

Fraction of the filtered water excreted as urine1

Urine flow rate, mL/kg per min

Effective renal plasma flow, mL/kg per min

Glomerular filtration rate, mL/kg per min

Variable

257 248 5.8 6.6

.0 .1

.2 .4

34.4 22.3

4.8 4.3

HC

TABLE 2. Kidney function values during CONTROL and Ca-LOADING urine collection periods, c nonnal (NC: 1%) or high (HO 3.5%) Ca alone or in combination with DL-methionine (DL

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69

HYPERCALCIURIC RESPONSE

loads (FECa) during Ca-LOADING than birds fed the HC basal diet. The CaLOADING period caused birds in the HCDL and HCAS groups to excrete more Ca in their urine than had entered the tubules through glomerular filtration, as indicated by fractional Ca excretion values substantially above 1.0 (net tubular Ca secretion). Dietary supplementation with DL-methionine and ammonium sulfate did not significantly affect fractional Ca excretion in groups fed 1% Ca. Groups fed the NCDL, NCAS, HCDL, and HCAS diets maintained significantly lower plasma Ca concentrations during CaLOADING than the respective NC and HC control groups (Table 3). Plasma P; concentrations were significantly depressed by Ca-LOADING in all diet treatment groups (Table 4) which, when combined with simultaneous reductions in glomerular filtration rate (Table 2), resulted in significant reductions in the filtered load of P; in all groups (Table 4). Among the groups fed 1% Ca, CaLOADING significantly reduced absolute (NC, NCDL, NCAS) and fractional (NCDL, NCAS) P; excretion. For birds fed 3.5% Ca, Ca-LOADING significantly reduced absolute P^ excretion only in the HCAS treatment group. Fractional Pj excretion was not significantly reduced by CALCIUM LOADING in groups fed 3.5% Ca diets. Dietary supplementation with DL-methionine and ammonium sulfate did not consistently affect plasma Pj or absolute and fractional P; excretion (Table 4). DISCUSSION In the present and previous studies, hypercalciuria, hypophosphaturia, and a significant increase in urine pH were observed when pullets were reared on 3.5% Ca diets, or when pullets reared on 1% Ca diets were infused intravenously with a Ca solution containing a metabolizable anion (Wideman et ah, 1985; Wideman, 1987; Glahn et al, 1988a, 1989). These results fully support the previously stated hypothesis that high dietary Ca:aP ratios cause hypercalciuric and hypophosphaturic responses similar to those observed following inhibition or removal of the

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supplemented with DL-methionine or ammonium sulfate (NCDL, NCAS) were compared with birds fed the basal NC diet (Table 3). Plasma Ca also was significantly lower during CONTROL or Ca-LOADING periods when birds fed the HCDL and HCAS diets were compared with birds fed the basal HC diet. Data presented in Tables 2 and 3 illustrate the interaction between glomerular filtration rate and plasma Ca concentrations in determining the filtered load of Ca. For example, during CONTROL urine collection periods, the NCAS group had the highest glomerular filtration rate (Table 2) but the lowest plasma Ca concentration (Table 3) of all groups fed 1% Ca diets, resulting in a filtered load of Ca that was not significantly different during CONTROL periods from the respective values of birds fed the NC or NCDL diets. Similar comparisons indicate the high filtered loads of Ca calculated for the NCAS and HC groups during Ca-LOADING (Table 3) primarily occurred because birds in these groups maintained relatively high glomerular filtration rates throughout intravenous Ca infusion (Table 2). During CONTROL urine collection periods, absolute and fractional Ca excretion rates for the separate treatment groups (Table 3) tended to reflect the overall impact of dietary Ca levels (Table 1); however dietary Ca did not determine the degree of calciuria observed during Ca-LOADING periods. Groups fed 1% Ca diets exhibited a range of Ca excretion rates (6.5 to 10.3 jtM/kg per min) that were very similar to the ranges (6.5 to 10.6 ftM/kg per min) exhibited by groups fed 3.5% Ca diets during Ca-LOADING (Table 3). Significant influences of DL-methionine and ammonium sulfate were not evident when the separate group means for absolute Ca excretion were compared during CONTROL periods, but DL-methionine and ammonium sulfate significantly influenced Ca excretion patterns during CaLOADING. Birds fed the NCAS and HCDL diets had significantly higher absolute rates of Ca excretion during CaLOADING than birds fed the respective NC or HC basal diets, and birds fed the HCDL and HCAS diets excreted significantly higher fractions of their filtered Ca

CONTROL Ca-LOADING CONTROL Ca-LOADING

CONTROL Ca-LOADING

CONTROL Ca-LOADING

Infusion protocol

± 1.9b ±2.0b

.2 ± .7b 6.5 .015 ± .013c .74 ± .22b

13.0 12.8

4.49 ± .41-1 8.88 ± .16a

NC

.1 7.5 .013 .53 ± .004c ± .07bc

± .02 d ± 1.4b

± 1.**

± 1.5<

.2 10.3 .016 .64

9.2 16.9 ± ± ± ±

.1«* 1.5" .004C .06b

± l.lbc + 2.1"

2.25 + .28* 6.40 ± .40 c

2.52 ± .43* 7.78 ± .70b 8.1 14.5

NCAS

NCDL

2.8 6.5 .47 .40

5.6 17.5

2.20 7.10

HC

CONTROL Ca-LOADING CONTROL Ca-LOADING

CONTROL Ca-LOADING 3.21 .19 .41 .28

± ± ± ± .55c .03e .04* .09c

8.0 ± 1.1b 1.5 ± .4*

1.52 ± .06 .48 ± .10*=

CONTROL Ca-LOADING

a

NC

Infusion protocol

.88 ± .10a .27 ± .07"*

7.38 ± .94" .43 ± .06 de

8.6 ± .9b 2.0 ± .3 e

1.52 ± .06" .58 ± .06d

NCDL ab

4.66 .20 .45 .09

± ± ± ±

.35b .02e .041* .01 d

10.5 ± .7" 2.8 ± .3 d e

1.45 ± .06 .58 ± .06"

NCAS

"Means (± SEM, n = 24) with no common superscript within a variable differ significantly (P < .05). *No units.

Fractional excretion of P;1

Urinary Pj excretion rate, pM/kg per min

Filtered load of Pj, /tM/kg per min

Total plasma Pj, mM

Variable

.68 .15 .10 .06

6.1 3.8

1.30 .82

HC

TABLE 4. Plasma inorganic phosphorus (Pj) and Pj excretion values during CONTROL and Ca-LOADING diets containing normal (NC: 1%) or high (HC: 3.5%) Ca alone or in combination with DL-methio

- Means (± SEM, n = 24) with no common superscript within a variable differ significantly (P :£ .05). 'No units.

a f

Fractional excretion of Ca1

Urinary Ca excretion rate, /tM/kg per min

Filtered load of Ca, pM/kg per min

Total plasma Ca, mM

Variable

TABLE 3. Plasma Ca and Ca excretion values during CONTROL and Ca-LOADING urine collection per normal (NC: 1%) or high (HC: 3.5%) Ca alone or in combination with DL-methionine (D

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HYPERCALCIURIC RESPONSE

nM. Pullets weighing 1.27 kg and excreting Ca at a rate of 2.10 jiM/kg per min (Table 1) would have an average Ca excretion rate of 2.67 /iM/min, which would clear all of the Ca, including the nonultrafilterable fraction, from the plasma within 81 min (216 fiM total plasma Ca/2.67 fiM Ca excreted/min). Similar calculations for pullets fed the NC diets (.14 fiM Ca excreted/kg per min; Table 1) yield a plasma Ca clearance rate of 1,215 min. Obviously some of the Ca excreted would be replenished from bone and extracellular fluid reserves; nevertheless high rates of urinary Ca excretion by birds fed HC diets may have significantly reduced plasma Ca concentrations prior to the collection of control plasma samples. A similar effect on plasma Na concentrations previously was noted for birds having high rates of urinary Na excretion (Wideman et al, 1987). In the future, this possibility can be addressed by collecting initial plasma samples prior to surgical preparation. As demonstrated previously using MHA (Wideman et al, 1989), dietary supplementation with DL-methionine and ammonium sulfate had little apparent impact on absolute or fractional Ca excretion during control clearance periods, although control plasma Ca concentrations were significantly lower for the NCDL and NCAS groups than for the NC group, and for the HCDL and HCAS groups than for the HC group. To the extent that Ca excretion during surgical preparations and equilibration may have contributed to the lower plasma Ca concentrations of groups fed diets supplemented with DLmethionine and ammonium sulfate, the consequent reduction in the filtered load of CA could mask any tendency toward increased absolute Ca excretion rates during control urine sample collections. If so, then increasing the filtered load of Ca by infusing Ca intravenously should reveal the latent calciuric efficacies of DLmethionine and ammonium sulfate. Calcium loading was effective in increasing plasma Ca concentrations, but the concurrent significant reductions in glomerular filtration rate tended to compromise the overall increase in the filtered Ca load for birds fed the NC, NCDL, and NCAS diets.

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parathyroid glands (Wideman, 1987; Wideman et al, 1989). The concurrent increase in urine pH apparently reflects the "alkalinizing" impact of the excess Ca on cation:anion balance, rather than a response to parathyroid inhibition (Laverty and Wideman, 1985; Clark and Wideman, 1989). Ingested or infused Ca should have a systemic alkalinizing influence when the accompanying anion is a metabolizable or "volatile" anion such as the carbonate moiety of calcium carbonate in the 3.5% Ca diet, or the acetate moiety of the Ca acetate infusion solution (Mongin, 1968). In contrast, the "fixed" sulfate anions derived from DL-methionine and ammonium sulfate would be predicted to have a systemic acidifying impact (Mongin, 1968). During CONTROL periods for example, DL-methionine and ammonium sulfate supplementation reduced the urine pH of the HCDL and HCAS groups from the more alkaline levels caused by the HC diet to levels that no longer were significantly higher than urine pH values of the NC, NCDL, and NCAS groups. The existing data fail to support one key aspect of the hypothesis that high dietary Ca:aP ratios cause parathyroid inhibition through hyperabsorptive hypercalcemia (Wideman et ah, 1989). Plasma Ca concentrations of birds reared on 3.5% Ca diets can be higher (Wideman et al, 1989) but often are equal to or lower than those of birds reared on 1% Ca diets (Wideman et at, 1985; Glahn et al, 1988a,b; Table 3). These observations suggest that diets having high Ca:aP ratios may cause parathyroid inhibition through elevated plasma 1,25-dihydroxycholecalciferol concentrations rather than through hypercalcemia (Henry et ah, 1977; Cantly et ah, 1987). Alternatively, plasma Ca concentrations of pullets reared on 3.5% Ca diets may be significantly depleted by high Ca excretion rates during the 60 to 120 min required for surgical preparation and equilibration. Assuming plasma volume averages 5.5% of body mass (.055 mL plasma/g body mass) in 8- to 16-wk-old pullets (Sturkie, 1986), and plasma Ca normally averages 3.09 mM/L = 3.09 /iM/ mL (Table 1, All NC groups), then the total plasma Ca content for pullets weighing 1,270 g (Table 1) would average 216

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study (200 mM Ca infused at a rate of .2 mL/kg per min = 40 fiM/kg per min) caused a significant reduction in glomerular filtration rate in pullets reared on 1% Ca diets (Table 2). These discrepancies may reflect the impact of differential volume expansion during the Ca-LOADING period, secondary to the use of infusion rates ranging from .1 to .4 mL/kg per min in different experiments. For example, glomerular filtration rate and effective renal plasma flow increase significantly when domestic fowl are volume expanded by increasing the intravenous infusion rate from .1 or .2 to .4 mL/kg per min (Wideman and Satnick, 1989; Wideman et al, 1989; Gregg and Wideman, 1990), and volume expansion may help attenuate the hypotension induced by high dietary Ca or Ca infusion (Lee et al, 1984; Koide and Tuan, 1989). Blood pressure was not recorded during the present study, but reductions in glomerular filtration rate would be expected if Ca infusion caused renal arterial perfusion pressure to drop below 70 mm Hg (Wideman, 1991; Wideman et al, 1992). The variable effects of dietary Ca and Ca-LOADING on glomerular filtration rate clearly emphasizes the need to measure glomerular filtration rate and calculate filtered load of Ca and fractional Ca excretion in experiments designed to assess the effects of dietary Ca:aP ratios or acid-base balance on renal tubular Ca transport. For example, absolute Ca excretion did not differ significantly among most treatment groups during control urine collections in the present study, but the filtered load of Ca was significantly lower and thus fractional Ca excretion was significantly higher in the HCAS and HCDL groups than in all of the NC groups (Table 3). Therefore, the combined effects of high dietary Ca and supplemental sulfur-containing compounds inhibited renal tubular Ca reabsorption and stimulated net tubular Ca secretion to the extent that several birds in the HCDL group excreted substantially more than 100% of their filtered loads of Ca, whereas birds in the NCDL and NCAS groups excreted less than 2% of their filtered loads of Ca. These observations clearly demonstrate that tubular Ca transport cannot be assumed to

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Nevertheless, as demonstrated in a previous study involving MHA (Wideman et al, 1989), Ca-LOADING effectively revealed significantly higher absolute rates of urinary Ca excretion for the NCAS and HCDL groups, and significantly higher fractional Ca excretion rates for the HCDL and HCAS groups. The latter response in particular demonstrates that DL-methionine and ammonium sulfate directly or indirectly altered tubular Ca transport, because fractional excretion calculations fully compensate for changes in the filtered load of Ca caused by changes in glomerular filtration rate or plasma Ca concentrations. Therefore, the increased fractional Ca excretion values for the HCDL and HCAS groups cannot be attributed to increased bone mineral solubilization or to an increased ultrafilterable fraction of plasma Ca caused by metabolic acidosis (Wideman et al, 1989). Instead, DLmethionine and ammonium sulfate may be calciuric because they tend to neutralize the anticalciuric impact of alkalosis induced by high dietary Ca:aP ratios, thereby fully revealing the hypercalciuria caused by parathyroid inhibition. Alternatively, acidifiers may have a more direct inhibitory influence on tubular Ca reabsorption or a stimulatory influence on tubular Ca secretion. In contrast to the highly consistent effects of dietary Ca or acute CaLOADING on urinary Ca, Pi7 and H+ excretion rates, the concurrent influences on glomerular filtration rate and effective renal plasma flow have been much less consistent across separate experiments. In independent studies using similar experimental techniques, diets having high Ca:aP ratios reduced or had no significant effect on glomerular filtration rate, and reduced or had no significant effect on effective renal plasma flow (Wideman et al, 1985, 1989; Glahn et al, 1988a, 1989; Table 2). In one previous study, the glomerular filtration rate increased transiently when pullets reared on 1% dietary Ca were infused with 100 mM Ca at a rate of .4 mL/kg per min, which represents an absolute Ca delivery rate of 40 /*M/kg per min (Wideman et al, 1989). Ca-LOADING at the same absolute rate in the present

HYPERCALCIURIC RESPONSE

REFERENCES Bottje, W. G., and P. C. Harrison, 1985. The effect of tap water, carbonated water, sodium bicarbonate, and calcium chloride on blood acid-base balance in cockerels subjected to heat stress. Poultry Sci. 64:107-113. Brun, C, 1957. A rapid method for determination of para-aminohippuric acid in kidney function tests. J. Lab. Clin. Med. 37:955-958. Cantley, L. K., J. B. Russell, D. S. Lettieri, and L. M. Sherwood, 1987. Effects of vitamin D3, 25-hydroxyvitamin D3, and 24,25-dihydroxyvitamin D3 on parathyroid hormone secretion. Calcif. Tissue Int. 41:48-51. Clark, N. B., and R. F. Wideman, 1989. Actions of parathyroid hormone and calcitonin in avian osmoregulation. Pages 111-125 in: Progress in Avian Osmoregulation. M. R. Hughes and A. Chadwick, ed. Leeds Philosophical and Literary Society (Scientific Section), Leeds, England. Fiske, C. H., and Y. Subbarow, 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-100. Frost, T. J., D. A. Roland, Sr., and D. N. Marple, 1991. The effects of various dietary phosphorus levels on the circadian patterns of plasma 1,25-dihydroxycholecalciferol, total calcium, ionized calcium, and phosphorus in laying hens. Poultry Sci. 70:1564-1570. Glahn, R. P., R. F. Wideman, and B. S. Cowen, 1988a. Effect of gray strain infectious bronchitis virus and high dietary calcium on renal function of single comb white leghorn pullets at 6, 10, and 18 weeks of age. Poultry Sci. 67:1250-1263. Glahn, R. P., R. F. Wideman, and B. S. Cowen, 1988b. Effect of dietary acidification and alkalinization on urolith formation and renal function in single comb white leghorn laying hens. Poultry Sci. 67:1694-1701. Glahn, R. P., R. F. Wideman, and B. S. Cowen, 1989. Order of exposure to high dietary calcium and gray strain infectious bronchitis virus alters renal function and the incidence of urolithiasis. Poultry Sci. 68:1193-1204. Gregg, C. M., and R. F. Wideman, 1990. Morphological and functional comparisons of normal and hypertrophied kidneys of adult domestic fowl

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have remained unaffected by an experimental protocol simply because the concentration of Ca in the urine ([Ca]J or the urinary Ca excretion rate (ECa = UFR x [Ca]J appears to remain relatively constant. Furthermore, these experiments emphasize the need to conduct CaLOADING studies to evaluate renal responses to hypercalcemia and an increased filtered load of Ca. The calciuric influence of sulfur-containing compounds may have the most significant impact on overall Ca balance during the postprandial influx of Ca from the diet.

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plasma flow: contribution of the renal portal system. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 160:663-669. Wideman, R. F., J. A. Closser, W. B. Roush, and B. S. Cowen, 1985. Urolithiasis in pullets and laying hens: role of dietary calcium and phosphorus. Poultry Sci. 64:2300-2307. Wideman, R. F., R. P. Glahn, W. G. Bottje, and K. R. Holmes, 1992. Use of a thermal pulse decay system to assess avian renal blood flow during reduced renal arterial perfusion pressure. Am. J. Physiol. 262:R90-R98. Wideman, R. F., W. B. Roush, J. L. Satnick, R. P. Glahn, and N. O. Oldroyd, 1989. Methionine hydroxy analog (free acid) reduced avian kid-

ney damage and urolithiasis induced by excess dietary calcium. J. Nutr. 119:818-828. Wideman, R. F., and J. L. Satnick, 1989. Physiological evaluation of diuresis in commercial broiler breeders. Br. Poult. Sci. 30:313-326. Wideman, R. F., J. L. Satnick, W. J. Mitsos, K. R. Bennett, and S. R. Smith, 1987. Effect of saline adaptation .and renal portal sodium infusion on glomerular size distributions and kidney function in domestic fowl. Poultry Sci. 66:348-356. Zemel, M. B., S. Schuette, M. Hegsted, and H. M. Linkswiler, 1981. Role of the sulfur-containing amino acids in protein-induced hypercalciuria in men. J. Nutr. 111:545-552. Downloaded from http://ps.oxfordjournals.org/ at Mount Allison University on June 13, 2015