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Maximization of Calcium and Phosphate in Neonatal Total Parenteral

Nutrition

Article  in  Pediatrics International · April 2018

DOI: 10.1111/ped.13579

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Pediatrics International (2018) 0, 1–5 doi: 10.1111/ped.13579

Original Article

Maximization of calcium and phosphate in neonatal total

parenteral nutrition

Hidehiko Maruyama,1 Jumpei Saito,2 Miki Nagai,2 Mai Mochizuki,2 Yoichi Ishikawa2 and Yushi Ito1
1
Division of Neonatology Center for Maternal–Fetal, Neonatal and Reproductive Medicine, and 2Department of Pharmacy,
National Center for Child Health and Development, Tokyo, Japan

Abstract Background: Appropriate calcium and phosphate supplementation is essential for bone growth in preterm infants.
Using Rehabix-K2TM (AY Pharmaceuticals, Tokyo, Japan) and Pleamin-P InjectionTM (Fuso Pharmaceutical Indus-
tries, Osaka, Japan) as the total parenteral nutrition (TPN) and amino acid solution, respectively, we investigated
ways of maximizing calcium and phosphate in the TPN solution.
Methods: Rehabix-K2, Pleamin-P, calcium gluconate, sodium phosphate, 50% glucose, and water were mixed in
varying proportions to create 16 formulations. Precipitation assessment was done three times for each of the 16 for-
mulations, and was based on the Japanese Pharmacopeia.
Result: Precipitation was observed 24 h after mixing when the calcium and phosphate were 60 mEq/L and
30 mmol/L or 80 mEq/L and 40 mmol/L, respectively. No precipitation was observed when the calcium and phos-
phate were 20 mEq/L and 10 mmol/L, respectively. Precipitation was observed once out of three times, when the
calcium and phosphate were 40 mEq/L and 20 mmol/L, respectively, and the amino acids were 2% and 3% (mean
pH, 6.13 and 6.26, respectively). No precipitation was observed, however, when the calcium and phosphate were
40 mEq/L and 20 mmol/L, respectively, and the amino acids were 0% and 1% (mean pH, 5.88 and 6.05,
respectively).
Conclusion: Not only the concentration of calcium and phosphate, but also the pH of the TPN solution, are crucial
factors for precipitation. Based on these results, a well-balanced TPN solution maximizing calcium and phosphate
availability will be able to be formulated.

Key words calcium, pH, phosphate, precipitation, total parenteral nutrition.

Appropriate calcium and phosphate supplementation is essen-
tial for bone growth in extremely low-birthweight infants
(ELBWI). The European Society of Paediatric Gastroenterol-
ogy, Hepatology and Nutrition (ESPGHAN) recommendation
is 2.6–6 mEq/kg/day for calcium and 1–2.3 mmol/kg/day for
phosphate, respectively.1 The ideal ratio of calcium/phosphate
(mEq/mmol) is 2.6–3.4 according to ESPGHAN1 or 2.4
according to the Australasian Neonatal Parenteral Nutrition
Consensus Group.2 Before establishing enteral nutrition, total
parenteral nutrition (TPN) is necessary. In the clinical setting,
the recommendations sometimes cannot be met due to water
restriction for ELBWI and the calcium and phosphate concen-
trations in the TPN solution.
Excessive calcium and phosphate in the TPN solution
will form a precipitate, which might result in catheter
obstruction or more serious complications such as
Correspondence: Hidehiko Maruyama, MD PhD, Division of
Neonatology, Center for Maternal–Fetal, Neonatal and Reproduc-
tive Medicine, National Center for Child Health and Develop-
ment, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan. Email:
maruyama-h@ncchd.go.jp
Received 15 January 2018; revised 22 March 2018; accepted
3 April 2018.
pulmonary vascular emboli.3–6 Several reports showed how
much calcium and phosphate could be tolerated in the TPN
solution,3,5,7 but each report used different kinds of TPN
solution, and so on, rendering the results somewhat incom-
patible with each other. In the present study Rehabix-K2TM
(AY Pharmaceuticals, Tokyo, Japan) was used as the TPN
solution, and Pleamin-P InjectionTM (Fuso Pharmaceutical
Industries, Osaka, Japan) as the amino acid solution. Reha-
bix-K2 includes dipotassium glycerophosphate, calcium glyc-
erophosphate, and calcium lactate. The calcium and
phosphate concentrations are 15 mEq/L and 20 mmol/L,
respectively. The glucose concentration is 21%, and the pH
is 4.8–5.8. Rehabix-K2 also includes buffers such as lactate,
acetate, and citrate. Pleamin-P contains 7.6% amino acid,
has a pH of 6.5–7.5 and also includes acetate as a buffer.
There are no data on the interaction of calcium and phos-
phate in Rehabix-K2 and Pleamin-P.
Figure 1 shows the concentration of calcium and phosphate
in the TPN solution based on unpublished internal data from
the present neonatal intensive care unit (NICU; H Maruyama,
unpubl. data, 2017). These TPN solutions were made for
ELBWI born in 2016, and the data spanned the period from
day 0 to day 30 (n = 20; 288 formulations). To avoid

© 2018 Japan Pediatric Society

2 H Maruyama et al.

precipitation empirically, we added either calcium gluconate

Glucose

10.7
10.4

10.8
10.8

10.8
10.8
10.5
9.2
10.9
10.2
10.2
9.2
(%)

9.9
9.5

9.6
9.4
or sodium phosphate to Rehabix-K2.
The aim of this study was to determine the maximum cal-
cium and phosphate balance in the TPN solution for ELBWI.

Estimated concentration

(%)
AA

0.9
2.0
3.0

1.0
1.9
3.0

1.0
2.1
2.9

0.9
2.0
2.9
0

0
Methods

Phosphate
(mmol/L)
Room temperature was measured, and Rehabix-K2, calcium

9.44
10.2
10.0

9.1
20.5
20.5
20.6
20.1
30.9
30.9

30.6
39.1
39.0
39.0
39.6
gluconate (8.5%), sodium phosphate (disodium phosphate and

30
sodium dihydrogen phosphate, 0.5 mmol/mL), Pleamin-P,
50% glucose, and water were prepared. The concentration of
glucose was fixed at 10% in the TPN solution. The concen-

(mEq/L)
Calcium

19.6
19.1
20.0
20.1
39.7
39.7
39.1
39.4

60.2
59.8
80.9
79.6
79.6
79.7
tration of calcium, phosphate, and amino acid were varied in

60
60
16 different formulations. The calcium and phosphate satura-
tion curves were L-shaped.5 With the ideal ratio of calcium
to phosphate as a reference, a point slightly to the right of

Water

7.5

3.5

4.5
this value was chosen for reasons of safety. The target con-

9
7
5
2

5
4
1
6

1
0

3
0
0
centration, the amount of each substance, and the estimated

glucose
concentration are shown in Table 1. The numbers 1–16 indi-

50%

1.3
cate the formulation numbers.

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
The ingredients were mixed with a stirrer. Calcium and
phosphate were added last. The pH of the solution was mea-

Pleamin-
PTM

2.5
5.5

2.5
5.5

2.5
5.5

2.5
5.5
sured with a pH measurement instrument (LAQUA, Horiba, Mixture amount (mL)

7
0

7
0

7
0

8
Kyoto, Japan). After centrifugation (23 1009g, 5 min), the
solutions were evaluated for precipitation by visual inspec-
tion3 and stored for 24 h in the same room. Afterwards, the
phosphate
Sodium

0.4
0.4
0.5
0.4
0.8
0.8
0.8
0.8
1.1
1.2
1.2
1.4
degree of precipitation was assessed again after centrifuga-
0
0
0
0
tion. We described the precipitation detection method based
on 6.06 Foreign Insoluble Matter Test for Injections of The
Japanese Pharmacopoeia 17th edition (from 1 April 2016):
gluconate
Calcium

clean the exterior of containers, and inspect against both a

0.6
0.6
0.7
0.6
1.6
1.6
1.8
1.5
2.6
2.6
2.7
2.5
3.6
3.8
3.8
Table 1 Target concentration, quantity of mixture, and estimated concentration

white and a black background for 5 s each time with the

4
unaided eyes at a position of light intensity of 2,000–3,750 lx
under a white light source. We also measured the temperature
Rehabix-

of the solution immediately after mixing and after 24 h.

K2TM
10
10
10
8
10
10
10
8
10
10
10
8
10
10
10
6
Room temperature was kept at the same temperature.
Glucose
(%)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
(%)
AA
Target concentration

0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
Phosphate
(mmol/L)
10
10
10
10
20
20
20
20
30
30
30
30
40
40
40
40

Fig. 1 Calcium vs phosphate concentration in (●) total par-

enteral nutrition solutions formulated for extremely low-birth-
(mEq/L)
Calcium

weight infants born at the present hospital in 2016 between 1

20
20
20
20
40
40
40
40
60
60
60
60
80
80
80
80
AA, amino acid.

January and 31 December, spanning the period from day 0 to

day 30 (n = 20; 288 formulations). (○) Target calcium and phos-
phate concentrations in the present study. Shaded area, recom-
mended calcium and phosphate balance according to the
Mixture

European Society of Paediatric Gastroenterology, Hepatology

ID no.

and Nutrition1 and the Australasian Neonatal Parenteral Nutrition

10
11
12
13
14
15
16

Consensus Group.2
1
2
3
4
5
6
7
8
9

© 2018 Japan Pediatric Society

 Calcium and phosphate in neonatal TPN 3

This procedure was done three times and the data are
expressed as mean  SD.
This study was approved by the institution ethics committee.
Results
The room temperature was between 25.0 and 26.5°C. The
solution temperature was between 23.0 and 25.6°C. Precipita-
tion occurred in some of the combinations (Table 2; Fig. 2).
Precipitation occurred when the concentration of calcium and
of phosphate was high, for example, when calcium and phos-
phate were 60 mEq/L and 30 mmol/L or 80 mEq/L and
40 mmol/L, respectively. When the concentration of calcium
and phosphate was low, for example, when calcium and phos-
phate were 20 mEq/L and 10 mmol/L, respectively, there was
no precipitation. Different results were obtained when the cal-
cium and phosphate concentration was 40 mEq/L and
20 mmol/L, respectively, in 2% or 3% amino acid. No precip-
itation was evident immediately after mixing, but precipitation
was observed 24 h after mixing once out of three times. When
the calcium and phosphate were 40 mEq/L and 20 mmol/L,
respectively, with the amino acid concentrations at 0% or 1%,
there was no precipitation.
The pH of the solution increased as Pleamin-P concentra-
tion increased. Different results were obtained when the cal-
cium and phosphate were 40 mEq/L and 20 mmol/L,
respectively: the mean pH of the solution with an amino acid
concentration of 0%, 1%, 2%, and 3% was 5.88, 6.05, 6.13,
and 6.26, respectively (Fig. 2).
Discussion
The factors associated with calcium and phosphate solubility
are reportedly the pH, amino acid concentration, organic acid,
glucose concentration, and temperature.4,8,9 Of these, pH is
the most important.3–5,7,10 Eggert et al. noted six variations in
the calcium–phosphate concentration curves in different pH
conditions.5 In general, phosphate takes the form of monobasic
H2PO4- and dibasic HPO42 in solution. Monobasic phosphate
is relatively soluble while dibasic phosphate is highly insolu-
ble.5 At pH 7.4, 80% of phosphate takes the dibasic form, and
20% takes the monobasic form. Dibasic phosphate binds easily
to calcium. Monobasic phosphate becomes more abundant as
the pH decreases. Therefore, the low pH of the TPN solution
enables higher amounts of calcium and phosphate to be pre-
sent in the same solution. The pH buffers in Rehabix-K2 and
Pleamin-P may play an important role in maintaining the pH.
Dunham et al. showed that the calcium–phosphate satura-
tion curves differed when the amino acid concentration was
1% or 2%.4 The data on pH were not shown. The amino acid
used was TrophAmineTM, which has a pH of 5.0–6.0. The
amino acid concentration affected the pH of the solution.
Other studies used different amino acid preparations such as
VaminolactTM (pH 5.2),10 which is acidic. Pleamin-P, used in
the present study, is neutral (pH 6.5–7.5). Thus, the results dif-
fered when the calcium and phosphate were 40 mEq/L and
20 mmol/L, respectively. No precipitation was observed when
the amino acid concentration was 0% or 1%, but precipitation
was observed when the amino acid concentration was 2% or
3%. The pH of the solution with an amino acid concentration
of 0%, 1%, 2%, and 3% was 5.88, 6.05, 6.13, and 6.26,
respectively, suggesting that an alkaline pH may lead to the
formation of a precipitate. Even if the amino acid concentra-
tion is the same, the pH of the solution changed a little
because of the different concentration of calcium and phos-
phate.
Organic salts such as organic calcium and organic phos-
phate contained in Rehabix-K2 are highly soluble3 and may
have important effects. Calcium gluconate, an organic cal-
cium, can be used, but sodium phosphate, which is not

Table 2 Precipitation results

Target concentration Immediately after mixing After 24 h

Calcium (mEq/L) Phosphate (mmol/L) AA (%) Glucose (%) pH (mean  SD) Precipitation Precipitation
1 20 10 0 10 5.31  0.026 – –
2 20 10 1 10 5.70  0.11 – –
3 20 10 2 10 5.97  0.071 – –
4 20 10 3 10 6.13  0.033 – –
5 40 20 0 10 5.88  0.054 – –
6 40 20 1 10 6.05  0.071 – –
7 40 20 2 10 6.13  0.096 – + (once out of 3 times)
8 40 20 3 10 6.26  0.045 – + (once out of 3 times)
9 60 30 0 10 5.94  0.042 + +
10 60 30 1 10 6.07  0.085 + +
11 60 30 2 10 6.04  0.086 + +
12 60 30 3 10 6.23  0.056 + +
13 80 40 0 10 5.96  0.062 + +
14 80 40 1 10 6.03  0.080 + +
15 80 40 2 10 6.06  0.079 + +
16 80 40 3 10 6.13  0.059 + +
AA, amino acid.

© 2018 Japan Pediatric Society

4 H Maruyama et al.
Fig. 2 Precipitation status vs calcium and phosphate concentra-
tion at amino acid concentration (a) 0%; (b) 1%; (c) 2%; and (d)
3%. (○) No precipitation immediately or at 24 h after mixing;
(M) no precipitation immediately, but precipitation occurred after
24 h [once out of three times]; (□)precipitation occurred immedi-
ately and at 24 h after mixing) with, mean pH of the solution.
organic, is the only available phosphate. Hence care should be
exercised in its use.
Calcium and phosphate are more soluble when combined
with a high concentration of glucose.11 In the present study,
the glucose concentration was fixed at 10% based on a previ-
ous study.4
Calcium and phosphate are also more soluble at low temper-
atures.5 Some NICU use ready-made TPN solutions, which
should be stored at a low temperature and warmed before use.
Some studies have proposed a protocol for this purpose.3,10 In
Japan, TPN solutions are made and used on the same day. The
room temperature in the NICU is maintained at 25°C. The pre-
sent study was conducted at almost the same temperature.
Some studies have investigated the stability of all-in-one
solutions, which contain fat.12,13 We, however, usually sepa-
rately prepare IntraliposTM 20% (pH 6.5–8.5; Otsuka Pharma-
ceutical, Tokushima, Japan) as the fat solution and provide the
TPN and fat solutions via the same route. The injection speed
of the fat solution is much slower than that of TPN. Theoreti-
cally, Intralipos, which is alkaline, may affect the pH of TPN
solution, but we were unable to find previous research
addressing this issue.
Precipitation takes some time to form.9,14 In the clinical
setting, TPN solution is given over 24 h. We therefore
assessed precipitation after 24 h and identified it in two differ-
ent mixtures despite the absence of precipitation immediately
after mixing.
For bone formation, well-balanced calcium and phosphate
supplementation is necessary.15 The recommended ratio of cal-
cium : phosphate is 2.4–3.4 mEq : 1 mmol.1,2 The present
study has shown that, at any amino acid concentration, calcium
and phosphate concentrations of 20 mEq/L and 10 mmol/L,
respectively, are safe. Even when the amino acid concentration
is ≤1%, a calcium and phosphate concentration of 40 mEq/L
and 20 mmol/L, respectively, is also safe. For example, a com-
bination of calcium 40 mEq/L, phosphate 13 mmol/L, and
amino acid 1% in a solution 100 mL/kg/day would produce
calcium 4 mEq/kg/day and phosphate 1.3 mmol/kg/day and a
© 2018 Japan Pediatric Society
calcium : phosphate ratio of 3.1 mEq : 1 mmol, which satisfies
the recommendations. If the total water intake were increased
in a manner appropriate for the cardiac and respiratory status,
calcium and phosphate could be given stably. One way to
avoid precipitation is to use different routes for amino acid and
other TPN solutions.
This study had several limitations. First, we assessed cal-
cium and phosphate precipitation via visual inspection as in a
previous report,7,16 while other reports used electronic devices
such as a particle counter.3,10 Second, the results obtained in
this study from mixing the ingredients cannot be applied
directly to clinical situations due to differences in the concen-
tration of glucose, amino acids, and so on. Nonetheless, the
present findings should prove helpful for formulating an opti-
mal TPN solution.
In conclusion, the factors that are important for precipita-
tion are not only the concentration of calcium and phosphate,
but also the pH of the TPN solution. Based on these results,
we were able to produce a well-balanced TPN solution that
maximized the availability of calcium and phosphate.
Disclosure
The authors declare no conflict of interest.
Author contributions
H.M. designed the initial study and J.S., M.N. and M.M. mod-
ified it. H.M., J.S., M.N. and M.M. performed experiments
and wrote the initial manuscript. Y.Is. and Y.It. advised on
and corrected the manuscript. All authors read and approved
the final manuscript.
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© 2018 Japan Pediatric Society